Cd19-specific redirected immune cells

ABSTRACT

Genetically engineered, CD19-specific redirected immune cells expressing a cell surface protein having an extracellular domain comprising a receptor which is specific for CD19, an intracellular signaling domain, and a transmembrane domain. Use of such cells for cellular immunotherapy of CD19 +  malignancies and for abrogating any untoward B cell function. In one embodiment, the immune cell is a T cell and the cell surface protein is a single chain scFvFc:ζ receptor where scFv designates the V H  and V L  chains of a single chain monoclonal antibody to CD19, Fc represents at least part of a constant region of an IgG 1 , and ζ represents the intracellular signaling domain of the zeta chain of human CD3. The extracellular domain scFvFc and the intracellular domain ζ are linked by a transmembrane domain such as the transmembrane domain of CD4. A method of making a redirected T cell expressing a chimeric T cell receptor by electroporation using naked DNA encoding the receptor.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to Provisional ApplicationSerial No. 60/246,117, filed Nov. 7, 2000, the disclosure of which isincorporated by reference.

BACKGROUND OF THE INVENTION

[0002] This invention relates to the field of genetically engineered,redirected immune cells and to the field of cellular immunotherapy ofB-cell malignancies, B-cell lymphoproliferative syndromes and B-cellmediated autoimmune diseases. The publications and other materials usedherein to illuminate the background of the invention or provideadditional details respecting the practice are incorporated byreference.

[0003] Approximately half of all hematopoietic stem cell transplantation(HSC) procedures performed in the United States are for the treatment ofhematologic malignancy [1]. The initial obstacles for successful HSCtransplantation were in large part due to inadequate treatmentmodalities for ameliorating regimen-related toxicities and forcontrolling opportunistic infections and graft-versus-host disease(GVHD) [2-5]. As supportive care measures have improved over the lastdecade, post-transplant disease relapse has emerged as the majorimpediment to improving the outcome of this patient population [6-10].The inability of maximally intensive preparative regimens combined withimmunologic graft-versus-tumor reactivity to eradicate minimal residualdisease is the mechanism of treatment failure in allogeneictransplantation while, in the autologous setting, tumor contamination ofthe stem cell graft can also contribute to post-transplant relapse [11].Targeting minimal residual disease early after transplantation is onestrategy to consolidate the tumor cytoreduction achieved withmyeloablative preparative regimens and purge, in vivo, malignant cellstransferred with autologous stem cell grafts. The utility of therapeuticmodalities for targeting minimal residual disease shortly following stemcell rescue is dependent on both a limited spectrum of toxicity and thesusceptibility of residual tumor cells to the modality's antitumoreffector mechanism(s). The successful elimination of persistent minimalresidual disease should not only have a major impact on the outcome oftransplantation for hematologic malignancy utilizing currentmyeloablative preparative regimens but may also provide opportunities todecrease the intensity of these regimens and their attendant toxicities.

[0004] The prognosis for patients with bcr-abl positive AcuteLymphoblastic Leukemia (ALL) treated with chemotherapy is poor andallogeneic transplantation has offered a curative option for manypatients when an appropriate donor was available. For example at theCity of Hope, 76 patients with bcr-abl positive ALL were treated withallogeneic Bone Marrow Transplantation (BMT) from a HLA matched donor.Of these patients, 26 were in first remission, 35 were transplantedafter first remission. The two year probability of disease free survivalwas 68% with a 10% relapse rate in those patients transplanted in firstremission whereas for those patients transplanted after first remission,the disease-free survival and relapse rate were 36% and 38%,respectively [12]. Post-transplant Polymerase Chain Reaction (PCR)screening of blood and marrow for bcr-abl transcript is under evaluationas a molecular screening tool for identifying early those transplantrecipients at high risk for later development of overt relapse [13,14].Patients for whom detectable p190 transcript was detected following BMThad a 6.7 higher incidence of overt relapse than PCR negative patients.The median time from the development of a positive signal to morphologicrelapse was 80-90 days in these patients. The identification of patientsin the earliest phases of post-transplant relapse affords theopportunity for making therapeutic interventions when tumor burden islow and potentially most amenable to salvage therapy.

[0005] Recent advances in the field of immunology have elucidated manyof the molecular underpinnings of immune system regulation and haveprovided novel opportunities for therapeutic immune system manipulation,including tumor immunotherapy. Evidence supporting the potential ofimmune-mediated eradication of residual tumor cells following allogeneictransplantation can be inferred by comparing the disparate relapse ratesbetween recipients of syngeneic and non-T cell depleted matched siblingtransplants. Patients with chronic myelogenous leukemia in chronic phase(CML-CP), acute myelogenous leukemia in first complete remission (1^(st)CR), and acute lymphoblastic leukemia in 1^(st) CR who received a marrowtransplant from a syngeneic donor had an actuarial probability ofrelapse at 3 years of 45%, 49%, and 41%, respectively, whereas the ratesfor recipients of a non-T depleted marrow transplant from an HLAidentical sibling for the same diseases were 12%, 20%, and 24%,respectively [15-17]. The reduction of relapse rates followingallogeneic bone marrow transplantation has been most significant inpatients who develop acute and/or chronic GVHD. Currently, efforts arefocused on developing strategies to selectively augment thegraft-versus-leukemia (GVL) response in order to reduce post-transplantrelapse rates without the attendant toxicities of augmented GVHD.

[0006] Studies in animal models have established that donorMHC-restricted CD8⁺ and CD4⁺ α/β⁺ T cells specific for minorhistocompatibility antigens encoded by polymorphic genes that differbetween the donor and recipient are the principle mediators of acuteGVHD and GVL [18-21]. Recently, patients with CML in chronic phase whorelapse after allogeneic BMT have been identified as a patientpopulation for whom the infusion of donor lymphocytes (DLI) successfullypromotes a GVL effect [22,23]. Complete response rates of approximately75% are achieved with DLI cell doses in the range of 0.25-12.3×10⁸mononuclear cells/kg [24]. Although the antitumor activity of donorlymphocyte infusion underscores the potential of cellular immunotherapyfor CML, the clinical benefit of DLI has not been generalizable to allforms of hematologic malignancy. Relapsed ALL is much less responsive toDLI with a reported CR rate of less than 20%; when tumor responses areobserved, they are typically associated with significant GVHD morbidityand mortality [25]. In order to increase the therapeutic ratio of DLI,genetic modification of donor lymphocytes to express a suicide gene isbeing evaluated as a strategy to permit the in vivo ablation of donorlymphocytes should toxicity from GVHD warrant this maneuver [26,27].Alternately, efforts are underway to identify genes encoding minorhistocompatibility antigens (mHA's) with restricted hematopoieticexpression that elicit donor antigen-specific T cell responses. Theisolation, ex vivo expansion, and re-infusion of donor-derived clonesspecific for these mHA's has the potential of selectively augmenting GVLfollowing allogeneic bone marrow transplantation [28-30].

[0007] Non-transformed B-cells and malignant B-cells express an array ofcell-surface molecules that define their lineage commitment and stage ofmaturation. These were identified initially by murine monoclonalantibodies and more recently by molecular genetic techniques. Expressionof several of these cell-surface molecules is highly restricted toB-cells and their malignant counterparts. CD20 is a clinically usefulcell-surface target for B-cell lymphoma immunotherapy with anti-CD20monoclonal antibodies. This 33-kDa protein has structural featuresconsistent with its ability to function as a calcium ion channel and isexpressed on normal pre-B and mature B cells, but not hematopoietic stemcells nor plasma cells [31-33]. CD20 does not modulate nor does it shedfrom the cell surface [34]. In vitro studies have demonstrated that CD20crosslinking by anti-CD20 monoclonal antibodies can trigger apoptosis oflymphoma cells [35,36]. Clinical trials evaluating the antitumoractivity of chimeric anti-CD20 antibody IDEC-C2B8 (Rituximab) inpatients with relapsed follicular lymphoma have documented tumorresponses in nearly half the patients treated, although the clinicaleffect is usually transient [37-40]. Despite the prolonged ablation ofnormal CD20⁺ B-cells, patients receiving Rituximab have not manifestedcomplications attributable to B-cell lymphopenia [41].Radioimmunotherapy with ¹³¹I-conjugated and ⁹⁰Y-conjugated anti-CD20antibodies also has shown promising clinical activity in patients withrelapsed/refractory high-grade Non-Hodgkins Lymphoma but hematopoietictoxicities from radiation have been significant, often requiring stemcell support [42].

[0008] Unlike CD20, CD19 is expressed on all human B-cells beginningfrom the initial commitment of stem cells to the B lineage andpersisting until terminal differentiation into plasma cells [43]. CD19is a type I transmembrane protein that associates with the complement 2(CD21), TAPA-1, and Leu13 antigens forming a B-cell signal transductioncomplex. This complex participates in the regulation of B-cellproliferation [44]. Although CD19 does not shed from the cell surface,it does internalize [45]. Accordingly, targeting CD19 with monoclonalantibodies conjugated with toxin molecules is currently beinginvestigated as a strategy to specifically deliver cytotoxic agents tothe intracellular compartment of malignant B-cells [46-48]. Anti-CD19antibody conjugated to blocked ricin and poke-weed antiviral protein(PAP) dramatically increase specificity and potency of leukemia cellkilling both in ex vivo bone marrow purging procedures and whenadministered to NOD-SCID animals inoculated with CD19⁺ leukemia cells[49]. In vitro leukemia progenitor cell assays have provided evidencethat the small percentage of leukemic blasts with the capacity forself-renewal express CD19 on their cell surface. This conclusion wasderived from the observations that leukemic progenitor activity isobserved exclusively in fresh marrow samples sorted for CD19 positivecells and is not observed in the CD19 negative cell population [50].Additionally B43-PAP treatment of relapsed leukemic marrow specimensablates progenitor cell activity while a PAP conjugated antibody with anirrelevant specificity had no such activity [51]. Systemicadministration of the CD19-specific immunotoxin B43-PAP is currentlyundergoing investigation in phase I/II clinical trials in patients withhigh risk pre-B ALL [52].

[0009] Despite the antitumor activity of monoclonal anti-CD20 andanti-CD19 antibody therapy observed in clinical trials, the high rate ofrelapse in these patients underscores the limited capacity of currentantibody-based immunotherapy to eliminate all tumor cells [53]. Incontrast, the adoptive transfer of tumor-specific T cells can result incomplete tumor eradication in animal models and a limited number ofclinical settings [54,55]. The ability of transferred T cells todirectly recognize and lyse tumor targets, produce cytokines thatrecruit and activate antigen non-specific antitumor effector cells,migrate into tumor masses, and proliferate following tumor recognitionall contribute to the immunologic clearance of tumor by T cells [56].Expression-cloning technologies have recently permitted the geneticidentification of a growing number of genes expressed by human tumors towhich T cell responses have been isolated [57,58]. To date leukemia andlymphoma-specific antigens have not been identified that are bothbroadly expressed by malignant B-cells and elicit T cell responses.Consequently, preclinical and clinical investigation has focused oncombining antibody targeting of tumors with T cell effector mechanismsby constructing bispecific antibodies consisting of CD20 or CD19 bindingsites and a binding site for a cell-surface CD3 complex epitope. Suchbispecific antibodies can co-localize leukemia and lymphoma targets withactivated T cells resulting in target cell lysis in vitro [59-61]. Thein vivo antitumor activity of such bispecific antibodies has beenlimited, however, both in animal models as well as in clinical practice[62]. The discrepancy between in vitro activity and in vivo effectlikely reflects the inherent limitations in antibody immunotherapycompounded by the obstacles associated with engaging T cells and tumorcells via a soluble linker in a manner that yields a persistent andfunctional cellular immune response [63].

[0010] The safety of adoptively transferring antigen-specific CTL clonesin humans was originally examined in bone marrow transplant patients whoreceived donor-derived CMV-specific T cells [56]. Previous studies havedemonstrated that the reconstitution of endogenous CMV-specific T cellresponses following allogeneic bone marrow transplantation (BMT)correlates with protection from the development of severe CMV disease[64]. In an effort to reconstitute deficient CMV immunity following BMT,CD8⁺ CMV-specific CTL clones were generated from CMV seropositiveHLA-matched sibling donors, expanded, and infused into sibling BMTrecipients at risk for developing CMV disease. Fourteen patients weretreated with four weekly escalating doses of these CMV-specific CTLclones to a maximum cell dose of 10⁹ cells/m² without any attendanttoxicity [65]. Peripheral blood samples obtained from recipients ofadoptively transferred T cell clones were evaluated for in vivopersistence of transferred cells. The recoverable CMV-specific CTLactivity increased after each successive infusion of CTL clones, andpersisted at least 12 weeks after the last infusion. However, long termpersistence of CD8⁺ clones without a concurrent CD4⁺ helper response wasnot observed. No patients developed CMV viremia or disease. Theseresults demonstrate that ex-vivo expanded CMV-specific CTL clones can besafely transferred to BMT recipients and can persist in vivo asfunctional effector cells that may provide protection from thedevelopment of CMV disease.

[0011] A complication of bone marrow transplantation, particularly whenmarrow is depleted of T cells, is the development of EBV-associatedlymphoproliferative disease [66]. This rapidly progressive proliferationof EBV-transformed B-cells mimics immunoblastic lymphoma and is aconsequence of deficient EBV-specific T cell immunity in individualsharboring latent virus or immunologically naive individuals receiving avirus inoculum with their marrow graft. Clinical trials by Rooney et al.have demonstrated that adoptively transferred ex-vivo expandeddonor-derived EBV-specific T cell lines can protect patients at highrisk for development of this complication as well as mediate theeradication of clinically evident EBV-transformed B cells [54]. Nosignificant toxicities were observed in the forty-one children treatedwith cell doses in the range of 4×10⁷ to 1.2×10⁸ cells/m².

[0012] Genetic modification of T cells used in clinical trials has beenutilized to mark cells for in vivo tracking and to endow T cells withnovel functional properties. Retroviral vectors have been used mostextensively for this purpose due to their relatively high transductionefficiency and low in vitro toxicity to T cells [67]. These vectors,however, are time consuming and expensive to prepare as clinical gradematerial and must be meticulously screened for the absence ofreplication competent viral mutants [68]. Rooney et al. transducedEBV-reactive T cell lines with the NeoR gene to facilitate assessment ofcell persistence in vivo by PCR specific for this marker gene [69].Riddell et al. have conducted a Phase I trial to augment HIV-specificimmunity in HIV seropositive individuals by adoptive transfer usingHIV-specific CD8⁺ CTL clones [70]. These clones were transduced with theretroviral vector tgLS⁺HyTK which directs the synthesis of abifunctional fusion protein incorporating hygromycin phosphotransferaseand herpes virus thymidine kinase (HSV-TK) permitting in vitro selectionwith hygromycin and potential in vivo ablation of transferred cells withgancyclovir. Six HIV infected patients were treated with a series offour escalating cell dose infusions without toxicities, with a maximumcell dose of 5×10⁹ cells/m² [70].

[0013] As an alternate to viral gene therapy vectors, Nabel et al. usedplasmid DNA encoding an expression cassette for an anti-HIV gene in aPhase I clinical trial. Plasmid DNA was introduced into T cells byparticle bombardment with a gene gun [71]. Genetically modified T cellswere expanded and infused back into HIV-infected study subjects.Although this study demonstrated the feasibility of using a non-viralgenetic modification strategy for primary human T cells, one limitationof this approach is the episomal propagation of the plasmid vector in Tcells. Unlike chromosomally integrated transferred DNA, episomalpropagation of plasmid DNA carries the risk of loss of transferredgenetic material with cell replication and of repetitive randomchromosomal integration events.

[0014] Chimeric antigen receptors engineered to consist of anextracellular single chain antibody (scFvFc) fused to the intracellularsignaling domain of the T cell antigen receptor complex zeta chain (ζ)have the ability, when expressed in T cells, to redirect antigenrecognition based on the monoclonal antibody's specificity [72]. Thedesign of scFvFc:ζ receptors with target specificities for tumorcell-surface epitopes is a conceptually attractive strategy to generateantitumor immune effector cells for adoptive therapy as it does not relyon pre-existing anti-tumor immunity. These receptors are “universal” inthat they bind antigen in a MHC independent fashion, thus, one receptorconstruct can be used to treat a population of patients with antigenpositive tumors. Several constructs for targeting human tumors have beendescribed in the literature including receptors with specificities forHer2/Neu, CEA, ERRB-2, CD44v6, and epitopes selectively expressed onrenal cell carcinoma [73-77]. These epitopes all share the commoncharacteristic of being cell-surface moieties accessible to scFv bindingby the chimeric T cell receptor. In vitro studies have demonstrated thatboth CD4⁺ and CD8⁺ T cell effector functions can be triggered via thesereceptors. Moreover, animal models have demonstrated the capacity ofadoptively transferred scFvFc:ζ expressing T cells to eradicateestablished tumors [78]. The function of primary human T cellsexpressing tumor-specific scFvFc:ζ receptors have been evaluated invitro; these cells specifically lyse tumor targets and secrete an arrayof pro-inflammatory cytokines including IL-2, TNF, IFN-γ, and GM-CSF[79]. Phase I pilot adoptive therapy studies are underway utilizingautologous scFvFc:ζ-expressing T cells specific for HIV gp120 in HIVinfected individuals and autologous scFvFc:ζ-expressing T cells withspecificity for TAG-72 expressed on a variety of adenocarcinomasincluding breast and colorectal adenocarcinoma.

[0015] Investigators at City of Hope have engineered a CD20-specificscFvFc:ζ receptor construct for the purpose of targeting CD20+ B-cellmalignancy [80]. Preclinical laboratory studies have demonstrated thefeasibility of isolating and expanding from healthy individuals andlymphoma patients CD8+ CTL clones that contain a single copy ofunrearranged chromosomally integrated vector DNA and express theCD20-specific scFvFc:zζ receptor [81]. To accomplish this, purifiedlinear plasmid DNA containing the chimeric receptor sequence under thetranscriptional control of the CMV immediate/early promoter and the NeoRgene under the transcriptional control of the SV40 early promoter wasintroduced into activated human peripheral blood mononuclear cells byexposure of cells and DNA to a brief electrical current, a procedurecalled electroporation [82]. Utilizing selection, cloning, and expansionmethods currently employed in FDA-approved clinical trials at the FHCRC,gene modified CD8+ CTL clones with CD20-specific cytolytic activity havebeen generated from each of six healthy volunteers in 15 separateelectroporation procedures [81]. These clones when co-cultured with apanel of human CD20+ lymphoma cell lines proliferate, specifically lysetarget cells, and are stimulated to produce cytokines.

[0016] It is desired to develop additional redirected immune cells and,in a preferred embodiment, redirected T cells, for treating B-cellmalignancies and B-cell mediated autoimmune disease.

SUMMARY OF THE INVENTION

[0017] In one aspect, the present invention provides geneticallyengineered T cells which express and bear on the cell surface membrane aCD19-specific chimeric T cell receptor (referred to herein as “CD19R”)having an intracellular signaling domain, a transmembrane domain (TM)and a CD19-specific extracellular domain (also referred to herein as“CD19-specific T cells”). The present invention also provides theCD19-specific chimeric T cell receptors, DNA constructs encoding thereceptors, and plasmid expression vectors containing the constructs inproper orientation for expression.

[0018] In a second aspect, the present invention provides a method oftreating a CD19⁺ malignancy in a mammal which comprises administeringCD19-specific T cells to the mammal in a therapeutically effectiveamount. In one embodiment, CD8⁺ CD19-specific T cells are administered,preferably with CD4⁺ CD19-specific T cells. In a second embodiment, CD4⁺CD19-specific T cells are administered to a mammal, preferably with CD8⁺cytotoxic lymphocytes which do not express the CD19-specific chimericreceptor of the invention, optionally in combination with CD8⁺CD19-specific redirected T cells.

[0019] In another aspect, the present invention provides a method ofabrogating any untoward B cell function in a mammal which comprisesadministering to the mammal CD19-specific redirected T cells in atherapeutically effective amount. These untoward B cell functions caninclude B-cell mediated autoimmune disease (e.g., lupus or rheumatoidarthritis) as well as any unwanted specific immune response to a givenantigen.

[0020] In another aspect, the present invention provides a method ofmaking and expanding the CD19-specific redirected T cells whichcomprises transfecting T cells with an expression vector containing aDNA construct encoding the CD19-specific chimeric receptor, thenstimulating the cells with CD19⁺ cells, recombinant CD19, or an antibodyto the receptor to cause the cells to proliferate. In one embodiment,the redirected T cells are prepared by electroporation. In a secondembodiment, the redirected T cells are prepared by using viral vectors.

[0021] In another aspect, the present invention provides a method oftargeting Natural Killer (NK) cells which express and bear on the cellsurface membrane a CD19-specific chimeric immune receptor having anintracellular signaling domain, a transmembrane domain (TM) and aCD19-specific extracellular domain.

[0022] In another aspect, the present invention provides a method oftargeting macrophage cells which express and bear on the cell surfacemembrane a CD19-specific chimeric immune receptor having anintracellular signaling domain, a transmembrane domain (TM) and aCD19-specific extracellular domain.

[0023] In another aspect, the present invention provides a method oftargeting neutrophils cells which express and bear on the cell surfacemembrane a CD19-specific chimeric immune receptor having anintracellular signaling domain, a transmembrane domain (TM) and aCD19-specific extracellular domain.

[0024] In another aspect, the present invention provides a method oftargeting stem cells which express and bear on the cell surface membranea CD19-specific chimeric immune receptor having an intracellularsignaling domain, a transmembrane domain (TM) and a CD19-specificextracellular domain.

[0025] In another aspect, the invention provides a CD-19-specificchimeric T-cell receptor comprising an intracellular signalling domain,a transmembrane domain and a CD19-specific extracellular domain.

[0026] In one embodiment, the CD19-specific chimeric T cell receptor ofthe invention comprises scFvFc:ζ, where scFvFc represents theextracellular domain, scFv designates the V_(H) and V_(L) chains of asingle chain monoclonal antibody to CD19, Fc represents at least part ofa constant region of an IgG₁, and ζ represents the intracellularsignaling domain of the zeta chain of human CD3.

[0027] In another embodiment, the CD19-specific chimeric T cell receptorof the invention comprises the scFvFc extracellular domain and the ζintracellular domain are linked by the transmembrane domain of humanCD4.

[0028] In another embodiment, the CD19-specific chimeric T cell receptorof the invention comprises amino acids 23-634 of SEQ ID NO:2.

[0029] In another aspect, the invention provides a plasmid expressionvector containing a DNA construct encoding a chimeric T-cell receptor ofthe invention in proper orientation for expression.

BRIEF DESCRIPTION OF THE FIGURES

[0030] FIGS. 1A-1C show the double-stranded DNA sequence and amino acidsequence for the CD19:zeta chimeric immunoreceptor of the presentinvention, SEQ ID NO.1 and show the source of the DNA segments found inthe chimeric immunoreceptor.

[0031]FIG. 2 is a schematic representation of the plasmidpMG-CD19R/HyTK.

[0032]FIG. 3 shows Western blot analyses which demonstrate theexpression of the CD19R/scFvFc:ζ chimeric receptor.

[0033]FIG. 4 is a graphical representation showing the antigen-specificcytolytic activity of T-cells expressing the CD19R/scFvFc:ζ chimericreceptor.

[0034]FIG. 5 is a graphical representation of the production ofinterferon-γ by T cells expressing the CD19R/scFvFc:ζ chimeric receptorthat are incubated in the presence of various cell lines expressingCD-19.

[0035] FIGS. 6A-E are graphical representations showing theantigen-specific cytolytic activity of CD19R/scFvFc:ζ chimeric receptorredirected T-cell clones.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention is directed to genetically engineered,redirected T cells and to their use for cellular immunotherapy of B-cellmalignancies, Epstein Barr Virus-related lymphoproliferative disorders,and B-cell mediated autoimmune diseases.

[0037] In one aspect, the present invention provides geneticallyengineered T cells which express and bear on the cell surface membrane aCD19-specific chimeric T cell receptor having an intracellular signalingdomain, a transmembrane domain and a CD19-specific extracellular domain(referred to herein as CD19-specific T cells). The extracellular domaincomprises a CD19-specific receptor. Individual T cells of the inventionmay be CD4⁺/CD8⁻, CD4⁻/CD8⁺, CD4⁻/CD8⁻ or CD4⁺/CD8⁺. The T cells may bea mixed population of CD4⁺/CD8⁻ and CD4⁻/CD8⁺ cells or a population of asingle clone. CD4⁺ T cells of the invention produce IL-2 whenco-cultured in vitro with CD19⁺ lymphoma cells. CD8⁺ T cells of theinvention lyse CD19⁺ human lymphoma target cells when co-cultured invitro with the target cells. The invention further provides theCD19-specific chimeric T cell receptors, DNA constructs encoding thereceptors, and plasmid expression vectors containing the constructs inproper orientation for expression.

[0038] In a preferred embodiment, CD19-specific redirected T cellsexpress CD19-specific chimeric receptor scFvFc:ζ, where scFv designatesthe V_(H) and V_(L) chains of a single chain monoclonal antibody toCD19, Fc represents at least part of a constant region of a human IgG₁,and ζ represents the intracellular signaling domain of the zeta chain ofhuman CD3. The extracellular domain scFvFc and the intracellular domainζ are linked by a transmembrane domain such as the transmembrane domainof CD4. In other embodiments, the human Fc constant region may beprovided by other species of antibody such as IgG₄ for example.

[0039] In a specific preferred embodiment, a full length scFvFc:ζ cDNA,designated SEQ ID NO.1 or “CD19R:zeta,” comprises the human GM-CSFreceptor alpha chain leader peptide, FMC63 V_(H), Gly-Ser linker, FMC63V_(L), human IgG₄ Fc, human CD4 TM, and human cytoplasmic zeta chain.“Chimeric TCR” means a receptor which is expressed by T cells and whichcomprises intracellular signaling, transmembrane and extracellulardomains, where the extracellular domain is capable of specificallybinding in an HLA unrestricted manner an antigen which is not normallybound by a T cell receptor in that manner. Stimulation of the T cells bythe antigen under proper conditions results in proliferation (expansion)of the cells and/or production of cytokines (e.g., IL-2) and/orcytolysis.

[0040] In another aspect, the present invention provides a method oftreating a CD19⁺ malignancy, lymphoproliferative disease or autoimmunedisease mediated in part by B-cells in a mammal which comprisesadministering CD19-specific redirected T cells to the mammal in atherapeutically effective amount. In one embodiment of this aspect ofthe invention, a therapeutically effective amount of CD8⁺ CD19-specificredirected T cells are administered to the mammal. The CD8⁺ T cells arepreferably administered with CD4⁺ CD19-specific redirected T cells. In asecond embodiment of this aspect of the invention, a therapeuticallyeffective amount of CD4⁺ CD19-specific redirected T cells areadministered to the mammal. The CD4⁺ CD19-specific redirected T cellsare preferably administered with CD8⁺ cytotoxic lymphocytes whichexpress the CD19-specific chimeric receptor of the invention.

[0041] In another aspect, the invention provides genetically engineeredstem cells which express on their surface membrane a CD19-specificchimeric T cell receptor having an intracellular signaling domain, atransmembrane domain and a CD19-specific extracellular domain.

[0042] In another aspect, the invention provides genetically engineerednatural killer(NK) cells which express on their surface membrane aCD19-specific chimeric T cell receptor having an intracellular signalingdomain, a transmembrane domain and a CD19-specific extracellular domain.

[0043] In yet another aspect, the invention provides geneticallyengineered macrophage which express on their surface membrane aCD19-specific chimeric T cell receptor having an intracellular signalingdomain, a transmembrane domain and a CD19-specific extracellular domain.

[0044] In another aspect, the present invention provides a method ofabrogating any untoward B cell function in a mammal which comprisesadministering to the mammal CD19-specific redirected T cells in atherapeutically effective amount. Untoward B-cell functions can includeB-cell mediated autoimmune disease (e.g., lupus or rheumatoid arthritis)as well as any unwanted specific immune response to a given antigen. Forexample, CD19-specific redirected T cells can be administered in amethod of immunosuppression prior to administering a foreign substancesuch as a monoclonal antibody or DNA or virus or cell in the situationwhere any immune response would decrease the effectiveness of theforeign substance.

[0045] In another aspect, the present invention provides a method ofmaking and expanding the CD19-specific redirected T cells whichcomprises transfecting T cells with an expression vector containing aDNA construct encoding the CD19-specific chimeric receptor, thenstimulating the cells with CD19⁺ cells, recombinant CD19, or an antibodyto the receptor to cause the cells to proliferate. According to thisaspect of the present invention, the method preferably stably transfectsand re-directs T cells using electroporation of naked DNA.Alternatively, viral vectors carrying the heterologous genes are used tointroduce the genes into T cells. By using naked DNA, the time requiredto produce redirected T cells can be significantly reduced. “Naked DNA”means DNA encoding a chimeric T cell receptor (TCR) contained in aplasmid expression vector in proper orientation for expression. Theelectroporation method of this invention produces stable transfectantswhich express and carry on their surfaces the chimeric TCR (cTCR).

[0046] In a preferred embodiment, the T cells are primary human T cells,such as human peripheral blood mononuclear cells (PBMC), which havepreviously been considered resistant to stable transfection byelectroporation of plasmid vectors. Preferred conditions include the useof DNA depleted of endotoxin and electroporation within about 3 daysfollowing mitogenic stimulation of T cells. Following transfection, thetransfectants are cloned and a clone demonstrating presence of a singleintegrated unrearranged plasmid and expression of the chimeric receptoris expanded ex vivo. The clone selected for expansion preferably is CD8⁺and demonstrates the capacity to specifically recognize and lyselymphoma target cells which express the target antigen. The clone isexpanded by stimulation with IL-2 and preferably another stimulant whichis specific for the cTCR.

[0047] In another embodiment, the T cells are expressed inimmortalized/transformed cells such as the T-cell tumor line TALL101,for example.

[0048] The invention is described herein primarily with reference to thespecific scFcFv:ζ construct and receptor of SEQ ID Nos: 1 and 2, but theinvention is not limited to that specific construct and receptor. Basedon the V_(H) and V_(L) sequences of the CD19-specific murine IgG1monoclonal antibody published by Nicholson et al., a scFv sequence wasconstructed de novo utilizing PCR [83]. The scFv portion can be replacedby any number of different CD19 binding domains, ranging from a minimalpeptide binding domain, to a structured CD19 binding domain from a phagelibrary, to antibody like domains using different methods to hold theheavy and light chain together. The arrangement could be multimeric suchas a diabody. The secreted form of the antibody forms multimers. It ispossible that the T cell receptor variant is also a multimer. Themultimers are most likely caused by cross pairing of the variableportion of the light and heavy chains into what has been referred to byWinters as a diabody.

[0049] The hinge portion of the construct can have multiple alternativesfrom being totally deleted, to having the first cysteine maintained, toa proline rather than a serine substitution, to being truncated up tothe first cysteine. The Fc portion can be deleted, although there isdata to suggest that the receptor preferably extends from the membrane.Any protein which is stable and dimerizes can serve this purpose. Onecould use just one of the Fc domains, e.g, either the C_(H)2 or C_(H)3domain.

[0050] Alternatives to the CD4 transmembrane domain include thetransmembrane CD3 zeta domain, or a cysteine mutated CD3 zeta domain, orother transmembrane domains from other transmembrane signaling proteinssuch as CD16 and CD8. The CD3 zeta intracellular domain was taken foractivation. The intracellular signaling domain of the chimeric receptorof the invention is responsible for activation of at least one of thenormal effector functions of the immune cell in which the chimericreceptor has been placed. The term “effector function” refers to aspecialized function of a differentiated cell. Effector function of a Tcell, for example, may be cytolytic activity or helper activityincluding the secretion of cytokines. Thus the term “intracellularsignaling domain” refers to the portion of a protein which transducesthe effector function signal and directs the cell to perform aspecialized function. While usually the entire intracellular signalingdomain will be employed, in many cases it will not be necessary to usethe entire chain. To the extent that a truncated portion of theintracellular signaling domain may find use, such truncated portion maybe used in place of the intact chain as long as it still transduces theeffector function signal. The term intracellular signaling domain isthus meant to include any truncated portion of the intracellularsignaling domain sufficient to transduce the effector function signal.Examples include the zeta chain of the T cell receptor or any of itshomologs (e.g., eta, delta, gamma or epsilon), MB1 chain, B29, Fc RIIIand Fc RI and the like. Intracellular signaling portions of othermembers of the families of activating proteins can be used, such asFcγRIII and FcεRI. See Gross et al. [84], Stancovski et al. [73], Moritzet al. [75], Hwu et al. [85], Weijtens et al. [79], and Hekele et al.[76], for disclosures of cTCR's using these alternative transmembraneand intracellular domains.

[0051] Cellular Immunotherapy using Redirected T cells

[0052] The strategy of isolating and expanding antigen-specific T cellsas a therapeutic intervention for human disease has been validated inclinical trials [86, 65, 87]. Initial studies have evaluated the utilityof adoptive T cell therapy with CD8⁺ cytolytic T cell (CTL) clonesspecific for cytomegalovirus-encoded antigens as a means ofreconstituting deficient viral immunity in the setting of allogeneicbone marrow transplantation and have defined the principles andmethodologies for T cell isolation, cloning, expansion and re-infusion[86]. A similar approach has been taken for controlling post-transplantEBV-associated lymphoproliferative disease. EBV-specific donor-derived Tcells have the capacity to protect patients at high risk for thiscomplication as well as eradicate clinically evident disease whichmimics immunoblastic B cell lymphoma [87]. These studies clearlydemonstrate that adoptively transferred ex vivo expanded T cells canmediate antigen-specific effector functions with minimal toxicities andhave been facilitated by targeting defined virally-encoded antigens towhich T cell donors have established immunity.

[0053] The application of adoptive T cell therapy as a treatmentmodality for human malignancy has been limited by the paucity ofmolecularly-defined tumor antigens capable of eliciting a T cellresponse and the difficulty of isolating these T cells from thetumor-bearing host. Consequently, initial cellular immunotherapy trialsutilizing autologous antitumor effector cells relied on antigennonspecific effector cells such as lymphokine activated killer (LAK)cells which had limited efficacy and pronounced toxicities [88, 89]. Inan attempt to enhance the tumor-specificity of infused effector cells,IL-2 expanded tumor-infiltrating lymphocytes (TIL) were evaluated [90].Responses to TIL infusions were sporadic due in part to theheterogeneous population of cells expanded with unpredictable antitumorspecificities. Patients with melanoma and renal cell carcinoma howeveroccasionally manifested striking tumor regressions following TILinfusions and tumor-specific MHC-restricted T cell clones have beenisolated from these patients. Recently, expression cloning technologieshave been developed to identify the genes encoding tumor antigensthereby facilitating the development of recombinant DNA-based vaccinestrategies to initiate or augment host antitumor immunity, as well as invitro culture systems for generating tumor-specific T cells from cancerpatients [91 ]. Clinical trials utilizing autologous tyrosinase-specificCTL for the treatment of melanoma are currently underway.

[0054] The inclusion of hematogenous malignancies as targets for T celltherapy is warranted based on the observed graft versus leukemia (GVL)effect observed in the setting of allogeneic BMT and the capacity ofdonor buffy coat infusions to have anti-leukemic activity [92]. Atpresent, it is clear that T cells present in the marrow graft mount aresponse to host minor histocompatibility antigens (mHA's) contributingto graft versus host disease and there is increasing evidence that theremay be T cell specificities for GVL that are distinct from those of GVHDon the basis of restricted tissue expression of a subset of mHA's [93].Nevertheless, the susceptibility of malignant B cells to CTL recognitionand lysis is well documented [94, 95]. Efforts to target B cell lymphomawith MHC-restricted CTL have focused on the lymphoma clone's idiotype asa tumor-specific antigen. Murine models have demonstrated that CTLresponses can be generated to immunoglobulin variable regions and thatlymphoma cells process and present these determinants for T cellrecognition [96, 97]. Although these strategies are potentiallytumor-specific, they are also patient specific thus making large scaleapplication difficult.

[0055] Endowing T cells with a desired antigen specificity based ongenetic modification with engineered receptor constructs is anattractive strategy since it bypasses the requirement for retrievingantigen-specific T cells from cancer patients and, depending on the typeof antigen recognition moiety, allows for targeting tumor cell-surfaceepitopes not available to endogenous T cell receptors. Studies to definethe signaling function of individual components of the TCR-CD3 complexrevealed that chimeric molecules with intracellular domains of the CD3complex's zeta chain coupled to extracellular domains which could becrosslinked by antibodies were capable of triggering biochemical as wellas functional activation events in T cell hybridomas [98]. Recentadvances in protein engineering have provided methodologies to assemblesingle chain molecules consisting of antibody variable regions connectedby a flexible peptide linker which recapitulate the specificity of theparental antibody [99, 100]. Several groups have now reported on thecapacity of chimeric single chain receptors consisting of anextracellular scFv and intracellular zeta domain to re-direct T cellspecificity to tumor cells expressing the antibody's target epitope;receptor specificities have included HER2/Neu, and less wellcharacterized epitopes on renal cell and ovarian carcinoma [72, 73, 75,79, 84, 85]. An idiotype-specific scfv chimeric TCR has been describedwhich recognizes the idiotype-expressing lymphoma cell's surfaceimmunoglobulin as its ligand [101]. Although this approach swaps a lowaffinity MHC-restricted TCR complex for a high affinity MHC-unrestrictedmoleculer linked to an isolated member of the CD3 complex, thesereceptors do activate T cell effector functions in primary human T cellswithout apparent induction of subsequent anergy or apoptosis [79].Murine model systems utilizing scFv:ζ transfected CTL demonstrate thattumor elimination only occurs in vivo if both cells and IL-2 areadministered, suggesting that in addition to activation of effectorfunction, signaling through the chimeric receptor is sufficient for Tcell recycling [76].

[0056] Although chimeric receptor re-directed T cell effector functionhas been documented in the literature for over a decade, the clinicalapplication of this technology for cancer therapy is only now beginningto be applied. Ex vivo expansion of genetically modified T cells tonumbers sufficient for re-infusion represents a major impediment forconducting clinical trials. Not only have sufficient cell numbers beendifficult to achieve, the retention of effector function following exvivo expansion has not been routinely documented in the literature.

[0057] Treatment of CD19⁺ Malignancies with CD19-Specific Redirected TCells

[0058] This invention represents the targeting of a B cell malignancycell-surface epitope with CD19-specific redirected T cells. Malignant Bcells are an excellent target for redirected T cells, as B cells canserve as immunostimulatory antigen-presenting cells for T cells [102].Cytokine production by the CD19-specific scFvFc:ζ expressing Jurkatclones when co-cultured with CD19⁺ B-cell malignancy does not requirethe addition of professional antigen presenting cells to culture orpharmacologic delivery of a co-stimulatory signal by the phorbal esterPMA. The function of the CD19R:zeta chimeric immunoreceptor in T cellswas first assessed by expressing this scFvFc:ζ construct in primaryhuman T cell clones. Clones secrete cytokines (IFN-γ, TNF-α, and gm-CSF)specifically upon co-culture with human CD19⁺ leukemia and lymphomacells. Cytokine production by CD19-specific clones can be blocked inpart by the addition to culture of the anti-CD19 specific antibodyHIB19. Anti-CD20 antibody Leu-16 does not block cytokine productionthereby demonstrating the specificity of the CD19R:zeta chimericimmunoreceptor for CD19 on the tumor cell surface. CD19R:zeta⁺ CD8⁺ CTLclones display high levels of cytolytic activity in standard 4-hrchromium release assays against human CD19⁺ leukemia and lymphoma celllines cell lines and do not kill other tumor lines that are devoid ofthe CD19 epitope. These preclinical studies support the anti-tumoractivity of adoptive therapy with donor-derived CD19R:zeta-expressing Tcell clones in patients that relapse following HLA-matched allogeneicbone marrow transplantation.

[0059] We have found that expansion of CD19 specific re-directed CD8⁺CTL clones with OKT3 and IL-2 routinely results in the generation ofgreater than 10⁹ cells over a period of approximately six weeks, andthat the clones retain their effector function following expansion, asshown by functional chromium release assay data. Our observation thatthe plasmid/scFvFc:ζ system can generate transfectants with disruptedplasmid sequence underscores the desirability of cloning transfectantsand expanding those clones demonstrating the presence of a singleunrearranged integrated plasmid, expression of the chimeric receptor,and the capacity to specifically recognize and lyse CD19⁺ lymphomatarget cells.

[0060] CD19 is not tumor-specific and adoptive transfer of cells withthis specificity is expected to kill the subset of non-transformed Bcells which express CD19. Although CD19 is not expressed byhematopoietic stem cells or mature plasma cells, this cross-reactivitymay exacerbate the humoral immunodeficiency of patients receivingchemotherapy and/or radiotherapy. Equipping T cells with a suicide genesuch as the herpes virus thymidine kinase gene allows for in vivoablation of transferred cells following adoptive transfer withpharmacologic doses of gancyclovir and is a strategy for limiting theduration or in vivo persistence of transferred cells [27].

[0061] CD19-specific chimeric receptor-expressing T cells of thisinvention can be used to treat patients with CD19⁺ B-cell malignanciesand B-cell mediated autoimmunie diseases, including for example, acutelymphoblastic leukemia. High relapse rates observed following autologoustransplantation for leukemia can be reduced with post-transplant in vivotreatment with adoptively transferred CD19-specific redirected T cellsto purge CD19⁺ leukemic stem cells. CD19-specific redirected T cells canbe used to treat lymphoma patients with refractory or recurrent disease.The CD19⁺ redirected T cells can be administered following myeloablativechemotherapy and stem cell rescue, when tumor burden and normal CD19⁺cell burden are at a nadir and when the potential of an immunologicresponse directed against the scFvFc:ζ protein is minimized.

[0062] Patients can be treated by infusing therapeutically effectivedoses of CD8⁺ CD19-specific redirected T cells in the range of about 10⁶to 10¹² or more cells per square meter of body surface (cells/m²). Theinfusion will be repeated as often and as many times as the patient cantolerate until the desired response is achieved. The appropriateinfusion dose and schedule will vary from patient to patient, but can bedetermined by the treating physician for a particular patient.Typically, initial doses of approximately 10⁹ cells/m² will be infused,escalating to 10¹⁰ or more cells/m². IL-2 can be co-administered toexpand infused cells post-infusion. The amount of IL-2 can about 10³ to10⁶ units per kilogram body weight. Alternatively or additionally, anscFvFc:ζ-expressing CD4⁺ T_(H1) clone can be co-transferred to optimizethe survival and in vivo expansion of transferred scFvFc:ζ-expressingCD8⁺ T cells.

[0063] The dosing schedule may be based on Dr. Rosenberg's publishedwork [88-90]or an alternate continuous infusion strategy may beemployed. CD19-specific redirected T cells can be administered as astrategy to support CD8⁺ cells as well as initiate/augment a DelayedType Hypersensitivity response against CD19⁺ target cells.

[0064] It is known that chimeric immune receptors are capable ofactivating target-specific lysis by phagocytes, such as neutrophils andNK cells, for example (103). Thus the present invention alsocontemplates the use of chimeric T-cell receptor DNA to transfect intonon-specific immune cells including neutrophils, macrophages and NKcells. Furthermore, the present invention contemplates the use ofchimeric T-cell receptor DNA to transfect stem cells prior to stem celltransplantation procedures.

[0065] The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.(104-121, e.g.).

EXAMPLES

[0066] The present invention is further detailed in the followingexamples, which are offered by way of illustration and are not intendedto limit the invention in any manner, Standard techniques well known inthe art or the techniques specifically described below are utilized.

Example 1 Construction of a scFvFc:ζ cDNA Incorporating the FMC63 V_(H)and V_(L) Sequences

[0067] Based on the V_(H) and V_(L) sequences of the CD19-specificmurine IgG1 monoclonal antibody published by Nicholson et al., a scFvsequence was constructed de novo utilizing PCR [83]. A full lengthscFvFc:ζ cDNA designated CD19R:zeta was constructed by PCR spliceoverlap extension and consists of the human GM-CSF receptor alpha chainleader peptide, FMC63 V_(H), Gly-Ser linker, FMC63 V_(L), human IgG₁ Fc,human CD4 TM, and human cytoplasmic zeta chain. The nucleotide sequenceof the construct and the resulting amino acid sequence are set forth intogether in FIGS. 1A-C or separately as SEQ ID Nos:1 and 2,respectively.

[0068] The CD19-specific scFvFc:ζ receptor protein is expressed inPrimary Human T cells. To determine whether the CD19-specific scFvFc:ζconstruct could be expressed as an intact chimeric protein, T cells weretransfected with the plasmid of Example 1 containing the CD19R.Linearized plasmid was electroporated under optimized conditions andstable transfectants selected by addition of hygromycin to cultures.Referring now to FIG. 3, there are shown the results of Western blotanalyses of T-cells transfected with the CD19R receptor in an expressionvector of the present invention. Using methods known in the art, wholecell lysates from mock transfectants (cells containing the pMG plasmidwithout CD19R: MTH7B9), T-cells transfected with CD19R (SG1D12) andT-cells transfected with an anti-CD20 chimeric receptor (AR2H6) wereexamined. GAM-AP is alakaline phosphatase conjugated goat anti-mouseIgG. This is the second step detection reagent in the western blot thatproduces a chemiluminescent. Western blot of whole cell lysates with ananti-zeta antibody probe shows both the endogenous zeta fragment and theexpected intact 66-kDa chimeric receptor protein is expressed in cellstransfected with a chimeric receptor but not in cells transfected withplasmid lacking the DNA constructs of the present invention. Flowcytometric analysis with anti-murine Fab and anti-human Fc specificantibodies further confinmed the cell-surface expression of theCD19R:zeta scFvFc:ζ on T cell transfectants.

Example 2 CD19-Specific Re-Directed Effector Functions of T CellsExpressing the FMC63 Chimeric Immunoreceptor

[0069] 2(A)-Cytokine Production by Chimeric T-Cells:

[0070] Referring now to FIG. 4, the function of the CD19R:zeta chimericimmunoreceptor in T cells was first assessed by expressing this scFvFc:ζconstruct in primary human T cell clones. Clones secrete cytokines(IFN-γ, TNF-α, and gm-CSF) specifically upon co-culture with human CD19⁺leukemia and lymphoma cells. Using techniques well known in the art andfurther described herein, chimeric T-cell clones were isolated whichexpressed the CD19R:zeta chimeric immunoreceptor. FIG. 4 shows theresults of incubation of various T-cell clones expressing therecombinant CD19:zeta chimeric immunoreceptor with CD-19 leukemmia celllines. 1873-CRL is a human CD19+/CD20− ALL cell line purchased fromATCC. DHL-4 is a human CD19+/CD20+ lymphoma cell line. Iona=lonomycinpurchased from Sigma. This chemical is a calcium ionaphore.PMA=Phorbal-12-myristate-13 acetate (Sigma). Iono+PMA when added to Tcells maximally activates them for cytokine production. Supernatant of Tcells incubated with these chemicals serves as a positive control formaximal cytokine production. The cytokine assays are performed by adding10⁶ responsder T cells with the indicated stimulator (if the stimulatoris a tumor cell it is added at 2×10⁵ per 24-well and is irradiated 8Krads). The wells are supplemented with culture media to a final volumeof 2 mls and incubated for 72 hrs at which time cell-free supernatantsare harvested and assayed by specific ELISA using R+D Systems Kits perthe manufactuer's instructions.

[0071] Cytokine production by CD19-specific clones can be blocked inpart by the addition to culture of the anti-CD19 specific antibodyHIB19. Anti-CD20 antibody Leu-16 does not block cytokine productionthereby demonstrating the specificity of the CD19R:zeta chimericimmunoreceptor for CD19 on the tumor cell surface.

[0072] Referring now to FIG. 5, there are shown the results ofincubation of the T-cell clone SG1D12 with various cell lines asdescribed, some of which express the CD19 antigen. The graph representsthe results of ELISA with antibody specific for IFN-γ. These resultsdemonstrate that T-cells expressing the chimeric receptor release IFN-γin the presence of CD-19 bearing cells.

[0073] (2B)-Antige-Specific Cytolytic Activity of Chimeric T-Cells:

[0074] Referring now to FIG. 6, there are shown the results of chromiumrelease assays for 5 different chimeric T-cell clones (C11; C12; E8; F1and F3). CD19R:zeta⁺ CD8⁺ CTL clones display high levels of cytolyticactivity in standard 4-hr chromium release assays against human CD19⁺leukemia and lymphoma cell lines SUPB15, JM-1 and 1873 and do not killother tumor lines that are devoid of the CD19 epitope. These preclinicalstudies support the initiation of clinical investigation to explore thesafety and anti-tumor activity of adoptive therapy with donor-derivedCD19R:zeta-expressing T cell clones in patients that relapse followingHLA-matched allogeneic bone marrow transplantation.

Example 3 Generation and Characterization of T Cell Clones forTherapeutic Use

[0075] All T cells administered are TCR α/β⁺ CD4⁻CD8⁺ scFvFc:ζ⁺T cellclones containing unrearranged chromosomally integrated plasmid DNA. Tcells are isolated from the peripheral blood of the transplantrecipient's HLA-matched marrow donor. Materials and methods employed toisolate, genetically modify, and expand CD8⁺ T cell clones from healthymarrow donors are detailed in Examples 4-8. T cell clones geneticallymodified to express the CD19R:zeta scFvFc:ζ chimeric immunoreceptor andHyTK are selected for:

[0076] a. TCRα/β⁺, CD4⁻, CD8⁺ surface phenotype as determined by flowcytometry.

[0077] b. Presence of a single copy of chromosomally integrated plasmidvector DNA as evidenced by Southern blot.

[0078] c. Expression of the scFvFc:ζ gene product as detected by Westernblot.

[0079] d. Specific lysis of human CD19⁺ cell lines in 4-hr chromiumrelease assays.

[0080] e. Dependence on exogenous IL-2 for in vitro growth.

[0081] f. Mycoplasma, fungal, bacterial sterility and endotoxin levels<5 EU/ml.

[0082] g. In vitro sensitivity of clones to ganciclovir.

Example 4 Materials for Isolating, Genetically Modifying and ExpandingCD8⁺ T Cell Clones from Healthy Marrow Donors for Therapeutic Use

[0083] 1. Culture Media and Media Supplements

[0084] Culture media used in the studies include RPMI 1640 HEPES (IrvineScientific, Irvine, Calif.) for all cell cultures. All media ispurchased in 0.5 liter bottles and meets current FDA guidelines for usein adoptive immunotherapy studies in humans. Supplements to the culturemedia include L-glutamine (BioWhittaker, Walkersville, Md.) and fetalcalf serum (Hyclone, Logan, Utah) heat inactivated at 56° C. for 30minutes. All reagents are shipped to CRB-3008, inspected, and stored at−20° C. or 4° C. as appropriate for the reagent.

[0085] 2. OKT3

[0086] Orthoclone OKT3 (Ortho) 1 mg/ml purchased from the City of HopePharmacy and aliquoted into sterile cryovials are stored at −20° C. inCRB-3008 until thawed for study subject T cell expansion.

[0087] 3. Interleukin 2

[0088] Pharmaceutical grade recombinant human Interleukin-2 (rhIL-2)(Proleukin) is supplied in vials containing 0.67 mg of lyophilized IL-2and having a specific activity of 1.5×10⁶ IU/mg protein. The lyophilizedrecombinant IL-2 is reconstituted with sterile water for infusion anddiluted to a concentration of 5×10⁴ units/ml. IL-2 is aliquoted intosterile vials and stored at −20° C. in CRB-3008. rhIL-2 for directpatient administration is dispensed per standard practice.

[0089] 4. Plasmid DNA

[0090] The plasmid CD19R/HyTK-pMG containing the CD19-specific scFvFc:ζcDNA and HyTK cDNA constructs is manufactured under GLP conditions.Ampules containing 100 μg of sterile plasmid DNA in 40 μl ofpharmaceutical water. Vector DNA is stored in a −70° C. freezer inCRB-3008.

[0091] 5. Hygromicin

[0092] The mammalian antibiotic hygromycin is used to select geneticallymodified T cells expressing the HyTK gene. Commercially availablehygromycin (Invivogen, San Diego, Calif.) is prepared as a sterilesolution of 100 mg/ml active drug and is stored at 4° C. in CRB-3008.

[0093] 6. EBV-Induced B Cell Lines

[0094] Lymphoblastoid cell lines (LCL) are necessary feeder cells for Tcell expansion and have been used for this purpose in FDA-approvedclinical adoptive therapy trials. An EBV-induced B cell line designatedTM-LCL was established from a healthy donor by co-culture of PBMC withsupernatants of the B95-8 cell line (American Type Culture Collections)in the presence of cyclosporin A. This cell line is currently being usedas an irradiated feeder cell by investigators at the Fred HutchinsonCancer Research Center (FHCRC) and City of Hope National Medical Center.This cell line has tested negative for adventitious microorganisms aswell as EBV production by cord blood transformation assay. Workingstocks of TM-LCL have been cyropreserved in CRB-3008 after transfer fromDrs. Greenberg and Riddell at the FHCRC. These stocks have been thawedand retested for bacterial, fungal and mycoplasma sterility. TM-LCLfeeder cells are irradiated to 8,000 cGy prior to co-culture with Tcells.

[0095] 7. Feeder PBMCs

[0096] Peripheral blood mononuclear cells (PBMC) isolated from the studysubject's marrow harvested by leukapheresis and transferred to CRB 3008in a collection bag is used as autologous feeder cells. PBMC from thedonor's apheresis product in excess of that quantity needed forestablishing T cell cultures is cyropreserved in ampules containing50×10⁶-100×10⁶ mononuclear cells in the CRB-3008 liquid nitrogen tank.

Example 5 Generation of CD8⁺ CTL Clones Genetically Modified to Expressthe CD19-specific scFvFc:ζ Receptor and HyTK

[0097] 1. Peripheral Blood Lymphocytes—Collection and Separation

[0098] Peripheral blood mononuclear cells (PBMC) are obtained from thestudy subject's designated marrow donor by leukapheresis at the City ofHope National Medical Center. The mononuclear cells are separated fromheparinized whole blood by centrifugation over clinical grade Ficoll(Pharmacia, Uppsula, Sweden). PBMC are washed twice in sterile phosphatebuffered saline (Irvine Scientific) and suspended in culture mediaconsisting of RPMI, 10% heat inactivated FCS, and 4 mM L-glutamine.

[0099] 2. Activation of PBMC

[0100] T cells present in patient PBMC are polyclonally activated byaddition to culture of Orthoclone OKT3 (30 ng/ml). Cell cultures arethen incubated in vented T75 tissue culture flasks in the studysubject's designated incubator. Twenty-four hours after initiation ofculture rhIL-2 is added at 25 U/ml.

[0101] 3. Genetic Modification of Activated PBMC

[0102] Three days after the initiation of culture PBMC are harvested,centrifuged, and resuspended in hypotonic electroporation buffer(Eppendorf) at 20×10⁶ cells/ml. 25 μg of plasmid DNA together with 400μl of cell suspension are added to a sterile 0.2 cm electroporationcuvette. Each cuvette is subjected to a single electrical pulse of250V/40μs delivered by the Multiporator (Eppendorf) then incubated forten minutes at room temperature. Following the RT incubation, cells areharvested from cuvettes, pooled, and resuspended in phenol red-freeculture media containing 25 U/ml rhIL-2. Flasks are placed in thepatient's designated tissue culture incubator. Three days followingelectroporation hygromycin is added to cells at a final concentration of0.2 mg/ml. Electroporated PBMC are cultured for a total of 14 days withmedia and IL-2 supplementation every 48-hours.

[0103] 4. Cloning of Hygromycin-Resistant T Cells

[0104] The cloning of hygromycin-resistant CD8⁺ CTL from electroporatedOKT3-activated patient PBMC is initiated on day 14 of culture. Cellsexpressing FvFc product are positively selected for using antibodies toFab and Fc and/or Protein A-FITC label using techniques well known inthe art. Following incubation of electroporated cells with Fab and Fcantibody or Protein A-FITC, cells expressing the FvFc are isolated byimmunogenetic beads or colummns or fluorescent activated cell sortingprocedures. Viable patient PBMC are added to a mixture of 100×10⁶cyropreserved irradiated feeder PBMC and 20×10⁶ irradiated TM-LCL in avolume of 200 ml of culture media containing 30 ng/ml OKT3 and 50 U/mlrhIL-2. This mastermix is plated into ten 96-well cloning plates witheach well receiving 0.2 ml. Plates are wrapped in aluminum foil todecrease evaporative loss and placed in the patient's designated tissueculture incubator. On day 19 of culture each well receives hygromycinfor a final concentration of 0.2 mg/ml. Wells are inspected for cellularoutgrowth by visualization on an inverted microscope at Day 30 andpositive wells are marked for restimulation.

[0105] 5. Expansion of Hygromycin-Resistant Clones with CD19 Re-DirectedCytotoxicity

[0106] The contents of each cloning well with cell growth and cytolyticactivity by screening chromium release assay are individuallytransferred to T25 flasks containing 50×10⁶ irradiated PBMC, 10×10⁶irradiated LCL, and 30 ng/ml OKT3 in 25 mls of tissue culture media. Ondays 1, 3, 5, 7, 9, 11, and 13 after restimulation flasks receive 50U/ml rhIL-2 and 15 mls of fresh media. On day 5 of the stimulation cycleflasks are also supplemented with hygromycin 0.2 mg/ml. Fourteen daysafter seeding cells are harvested, counted, and restimulated in T75flasks containing 150×10⁶ irradiated PBMC, 30×10⁶ irradiated TM-LCL and30 ng/ml OKT3 in 50 mls of tissue culture media. Flasks receiveadditions to culture of rhIL-2 and hygromycin as outlined above.

[0107] 6. Characterization of Hygromycin-Resistant CTL Clones

[0108] a. Cell Surface Phenotype

[0109] CTL selected for expansion for use in therapy are analyzed byimmunofluorescence on a FACSCalibur housed in CRB-3006 usingFITC-conjugated monoclonal antibodies WT/31 (αβTCR), Leu 2a (CD8), andOKT4 (CD4) to confirm the requisite phenotype of clones (αβTCR⁺, CD4⁻,and CD8⁺). Criteria for selection of clones for clinical use includeuniform TCR αβ⁺, CD4⁻, CD8⁺ as compared to isotype controlFITC-conjugated antibody.

[0110] b. Chromosomal Integration of Plasmid

[0111] A single site of plasmid vector chromosomal integration isconfirmed by Southern blot analysis. DNA from genetically modified Tcell clones is screened with a DNA probe specific for the plasmidvector. The Hygro-specific DNA probe is the 420 basepair MscI/NaeIrestriction fragment isolated from CD19RR/HyTK-pMG. Probe DNA is ³²Plabeled using a random primer labeling kit (Boehringer Mannheim,Indianapolis, Ind.). T cell genomic DNA is isolated per standardtechnique. Ten micrograms of genomic DNA from T cell clones is digestedovernight at 37° C. with 40 units of XbaI and HindIII and thenelectrophoretically separated on a 0.85% agarose gel. DNA is thentransferred to nylon filters (BioRad, Hercules, Calif.) using analkaline capillary transfer method. Filters are hybridized overnightwith the HyTK-specific 32P-labeled probe in 0.5 M Na₂PO₄, pH 7.2, 7%SDS, containing 10 μg/ml salmon sperm DNA (Sigma) at 65° C. Filters arethen washed four times in 40 mM Na₂PO₄, pH 7.2, 1% SDS at 65° C. andthen visualized using a phosphoimager (Molecular Dynamics, Sunnyvale,Calif.). Criteria for clone selection is a single unique band with theHygro probe.

[0112] c. Expression of the CD19-Specific scFvFc:ζ Receptor

[0113] Expression of the CD19R scFvFc:ζ receptor is determined byWestern blot procedure in which chimeric receptor protein is detectedwith an anti-zeta antibody. Whole cell lysates of transfected T cellclones are generated by lysis of 2×10⁷ washed cells in 1 ml of RIPAbuffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) containing 1tablet/10 ml Complete Protease Inhibitor Cocktail (Boehringer Mannheim).After an eighty minute incubation on ice, aliquots of centrifuged wholecell lysate supernatant are harvested and boiled in an equal volume ofloading buffer under reducing conditions then subjected to SDS-PAGEelectrophoresis on a precast 12% acrylamide gel (BioRad). Followingtransfer to nitrocellulose, membranes are blocked in blotto solutioncontaining 0.07 gm/ml non-fat dried milk for 2 hours. Membranes arewashed in T-TBS (0.05% Tween 20 in Tris buffered saline pH 8.0) thenincubated with primary mouse anti-human CD3ζ monoclonal antibody 8D3(Pharmingen, San Diego, Calif.) at a concentration of 1 μg/ml for 2hours. Following an additional four washes in T-TBS, membranes areincubated with a 1:500 dilution of goat anti-mouse IgG alkalinephosphatase-conjugated secondary antibody for 1 hour. Prior todeveloping, membranes are rinsed in T-TBS then developed with 30 ml of“AKP” solution (Promega, Madison, Wis.) per the manufacturer'sinstructions. Criteria for clone selection is the presence of a 66 kDachimeric zeta band.

[0114] d. Cytolytic Specificity for CD19⁺ Leukemic Cells and Lack ofCytolytic Activity Against Recipient Fibroblasts Acitivity

[0115] CD8⁺ cytotoxic T cell clones expressing the CD19R scFvFc:ζreceptor recognize and lyse human CD19⁺ leukemia target cells followinginteraction of the chimeric receptor with the cell surface targetepitope in a HLA unrestricted fashion. The requirements for target cellCD19 expression and class I MHC independent recognition were confirmedby assaying several αβTCR⁺, CD8⁺, CD4⁻, CD19R⁺ CTL clones against apanel of MHC-mismatched human leukemia cell lines (SupB15, JM-1, and1873 CRL ) as well as the CD19⁻ line K562 (a CD19-negative, NK-sensitivetarget) and recipient fibroblasts. T cell effectors are assayed 12-14days following stimulation with OKT3. Effectors are harvested, washed,and resuspended in assay media; 2.5×10⁵, 1.25×10⁵, 0.25×10⁵, and0.05×10⁵ effectors are plated in triplicate at 37° C. for 4 hours with5×10³ target cells in V-bottom microtiter plates (Costar, Cambridge,Mass.). After centrifugation and incubation, 100 μL aliquots ofcell-free supernatant is harvested and counted. Percent specificcytolysis is calculated as follows:$\frac{\left( {{Experimental}\quad {\,^{51}{Cr}}\quad {release}} \right) - \left( {{control}\quad {\,^{51}{Cr}}\quad {release}} \right)}{\left( {{Maximum}\quad {\,^{51}{Cr}}\quad {release}} \right) - \left( {{control}\quad {\,^{51}{Cr}}\quad {release}} \right)} \times 100$

[0116] Control wells contain target cells incubated in assay media.Maximum ⁵¹Cr release is determined by measuring the ⁵¹Cr content oftarget cells lysed with 2% SDS. Criteria for clone selection is >50%specific lysis of both neuroblastoma targets at an effector:target ratioof 25:1 and less than 10% specific lysis of K562 and fibroblasts at anE:T ratio of 5:1.

Example 6 Microbiologic Surveillance of T Cell Cultures

[0117] Aliquots of media from the T cell cultures are screened by U.S.P.and fungal culture prior to cryopreservation (Stage I SterilityTesting). Cultures with evident contamination are immediately discarded.T cell expansions for re-infusion have U.S.P. and fungal cultures sent48-hrs prior to use (Stage II Sterility Testing). To detect mycoplasmacontamination, aliquots are assayed using the Gen-Probe test kit (SanDiego, Calif.) prior to cryopreservation (Stage I) and cultures withmycoplasma contamination discarded. Within 49-hrs of re-infusioncultures are re-screened as detailed above (Stage II). Prior to cellwashing on the day of re-infusion Gram stains are conducted on each bagto exclude overt contamination and endotoxin levels determined by LALare determined on the washed cell product. An endotoxin burden of <5EU/kg burden of endotoxin is allowed. Washed T cell clones are alsocyropreserved in case archival specimens are needed.

Example 7 Quality Control Criteria for Release of Clones for Re-Infusion

[0118] The criteria set forth in Table I must be met prior to release ofT cells for re-infusion. TABLE 1 Criteria for Release of Clones Testfor: Release Criteria: Testing Method: Viability of ClinicalPreparation >90% Trypan blue exclusion Cell-Surface Phenotype UniformlyTCRα/β⁺, Flow cytometric evaluation with CD4⁺, CD8⁺ isotype controls.Vector Integration Number Single band Southern Blot with Hygro- SpecificProbe scFvFc: ζ Expression 66-kD Band Western Blot with Human Zeta-Specific Primary Antibody CD19-Specific Anti-Leukemia >50% SpecificLysis at E:T 4 hr-Chromium Release Assay Cytolytic Ratio of 25:1 AgainstSUP-B19 Activity and JM-1 and <10% SL against K562 and fibros at an E:Tof 5:1. Sterility All Stage I U.S.P./fungal Bacterial/fungal by routinecultures neg at 21 days days. clinical specimen culture. Mycoplasma negat time of Mycoplasma by Gene-Probe cyropreservation and within RIA. 48hrs of each infusion. Endotoxin by LAL. Endotoxin level <5 E.I./kg inGram stain by clinical washed cell preparation. Gram microbiology lab.stain negative on day of re- infusion.

Example 8 Quantitative PCR For T Cell Persistence In Vivo

[0119] The duration of in vivo persistence of scFvFc:ζ⁺ CD8⁺ CTL clonesin the circulation is determined by quantitative PCR (Q-PCR) utilizingthe recently developed TaqMan fluorogenic 5′ nuclease reaction. Q-PCRanalysis is performed by the Cellular and Molecular Correlative Core ongenomic DNA extracted from study subject PBMC obtained prior to and ondays +1 and +7 following each T cell infusion. Following the thirdinfusion PBMC are also sampled on day +14, +21, +51 (Day +100 followingstem cell rescue). Should any study subject have detectablegene-modified T cells on day +100, arrangements are made to re-evaluatethe patient monthly until the signal is undetectable. Published datafrom Riddell et al. has determined that adoptively transferred T cellsare detected in the peripheral blood of study subjects one day followinga cell dose of 5×10⁹ cells/m² at a frequency of 1-3 cells/100 PBMC, thusthe doses of cells for this study will result in a readily detectablesignal (70). DNA is extracted from PBMC using the Qiagen QiAmp kit. Theprimers used to detect the scFvFc:ζ gene are 5′HcFc(5′-TCTTCCTCTACACAGCAAG CTCACCGTGG-3′; SEQ ID NO:3) and 3′HuZeta(5′-GAGGGTTCTTCCTTCTCG GCTTTC-3′; SEQ ID NO:4) and amplify a 360basepair fragment spanning the Fc-CD4-TM-zeta sequence fusion site. TheTaqMan hybridization probe is FAM-5′TTCACTCTGAA GAAGATGCCTAGCC3′-TAMRA(SEQ ID NO:5). A standard curve is generated from genomic DNA isolatedfrom a T cell clone with a single copy of integrated plasmid spiked intounmodified T cells at frequencies of 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, and 10⁻⁶. Acontrol primer/probe set specific for the human beta-globin gene is usedto generate a standard curve for cell number and permits the calculationof the frequency of genetically modified clone in a PBMC sample. Thebeta-globin amplimers are as follows: Pco3 (5′-ACACAACTGTGTTCACTAGC-3′;SEQ ID NO:6), GII (5′-GTCTCCTT AAACCTGTCTTG-3′; SEQ ID NO:7) and theTaqman probe is HEX-5′ACCTGACTCCTGAGG AGAAGTCT3′-TAMRA (SEQ ID NO:8).All patients will have persistence data and immune response data to thescFvFc:ζ and HyTK genes compared to determine if limited persistence canbe attributed to the development of an immune response to gene-modifiedT cells.

Example 9 Pilot Phase I Study

[0120] 1. Staging Criteria and Patient Eligibility

[0121] a. Staging Criteria

[0122] Prior to Study Enrollment

[0123] Immunohistopathologically confirmed CD19⁺ ALL.

[0124] Molecularly confirmed Ph⁺ ALL (Cohort 2)

[0125] Bone marrow aspirate and biopsy.

[0126] Lumbar puncture.

[0127] CT scan Chest/ABD/Pelvis.

[0128] After Study Enrollment

[0129] Donor Leukapheresis

[0130] Study Subject Skin Biopsy to Establish Fibroblast Cell Line

[0131] b. Patient Eligibility

[0132] Patient Inclusion Criteria

[0133] Relapsed CD19+ ALL following HLA-matched related donor BMT(Cohort 1).

[0134] History of Ph⁺ CD19⁺ ALL for which an HLA-matched allogeneic bonemarrow transplant is indicated (Cohort 2).

[0135] Male or female subjects ≧12 months of age and ≦65 years of age.

[0136] Consenting related donor HLA-phenotypically identical with thepatient for HLA-A, and -B and identical for DRB 1 alleles. Matchingassessed minimally by serology for Class I and DNA typing for Class IIantigens.

[0137] Patients with adequate organ function as measured by:

[0138] Cardiac: Asymptomatic or, if symptomatic, then left ventricularejection fraction at rest must be ≧50% or within normal range for COH.

[0139] Hepatic: SGOT within 5× normal range and total bilirubin <5×normal range.

[0140] Renal: Serum creatinine within 1.5× normal range or creatinineclearance 60 ml/min.

[0141] Pulmonary: DLCO >45% of predicted (corrected for hemoglobin) orwithin normal range for COH.

[0142] Adequate performance status 70% (Karnofsky-age >18yrs, Lansky≦18yrs of age).

[0143] Written informed consent from patient and donor conforming to COHguidelines obtained from donor and patient or patient & parent/legalguardian age (≧7yrs)

[0144] Life expectancy >8 weeks and absence of co-existingmedical/psychological problems which would significantly increase therisk of the transplant procedure and T cell re-infusions based on thejudgement of the study chairperson.

[0145] Availability of patient for peripheral blood sample drawing forstudy tests following transplantation as outlined in Appendix C.

[0146] Patient Exclusion Criteria

[0147] Prior autologous or allogeneic bone marrow or PBSC transplant(Cohort 2).

[0148] Patients who cannot complete total body irradiation doserequirements due to prior radiation treatment (Cohort 2).

[0149] Female patients who are pregnant or breast feeding.

[0150] Positive serology for HIV.

[0151] Active infection requiring intravenous treatment withanti-fungal, anti-bacterial or antiviral agents within two weeks priorto conditioning with the exception of coagulase negative staphylococcalline infection (Cohort2).

[0152] Failure to understand the basic elements of the protocol and/orthe risks/benefits of participating in this phase I study (Children≧7-yrs as well as parent/legal guardian as determined by performance ona questionnaire administered prior to consent signing).

[0153] Donor Selection

[0154] Willingness to undergo leukapheresis for PBMC collection.

[0155] 2. Study Design and Rules for Dose Escalation

[0156] The pilot Phase I study is an open-label, nonrandomized study. Inthis study patients either who suffer a relapse of their CD19⁺ ALLfollowing BMT (Cohort 1) or who experience a molecular post-transplantrelapse of their Ph⁺ CD19⁺ ALL receive donor-derived CD19R⁺HyTK⁺CD8⁺ CTLclones. T cell clones are generated from a leukapheresis productobtained from the patient's HLA-matched related marrow donor. Forpatients enrolled into Cohort 2, clones are cryopreserved until suchtime that the research participant is diagnosed with a molecular relapsebased on a positive and confirmatory PCR result for bcr-abl. Eachresearch participant in each cohort receives a series of threeescalating cell dose T cell infusions at two-week intervals beginning assoon as clones are available (typically by the 14th day following thediagnosis of molecular relapse in cohort 2, and as soon as clones areready in cohort 1). Those research subjects on immunosuppressivemedications for GVHD prophylaxis/treatment are first tapered offcorticosteroids and have no more than grade 2 AGVHD prior to commencingwith T cell administrations. The first cell dose is 1×10⁹ cells/m², thesecond 5×10⁹ cells/m², and the third 5×10⁹ cells/m² with IL-2. Patientswithout significant toxicity attributed to the T cell infusions and whohave ≦grade 2 GVHD receive low-dose s.c. rhIL-2 for 14 days with thethird T cell dose. Patients are evaluated prior to and weekly after thefirst infusion for a period of two months after which time, patients areevaluated monthly for an additional six months. Peripheral blood isdrawn at specific times during the study to assay for the in vivopersistence of the transferred CTL clones and the induction ofanti-scFvFc:ζ and HyTK immune responses. Anti-tumor responses areassessed by changes in the molecular tumor burden by serial Q-PCR fortheir leukemia-specific marker or bcr-abl, and, by standard morphologic,flow cytometric, and chimerism studies for ALL. The patient's primaryHematologist or pediatric oncologist manages the non-study specificaspects of their patient's medical management throughout the duration ofthe study and indefinitely thereafter.

[0157] 3. Treatment Plan

[0158] a. Schedule of Administration of CD19R:zeta⁺, CD8⁺ T Cell Clones

[0159] The phase I pilot study determines the safety and toxicity ofintravenously infused donor-derived CD8⁺ CTL clones genetically modifiedto express the CD19R scFvFc:ζ chimeric immunoreceptor and theselection/suicide gene HyTK. A series of three escalating cell doseinfusions (Table 2) are administered at two-week intervals to researchparticipants who demonstrate a post-transplant molecular relapse. T cellinfusions commence at the earliest time of their availability (Cohort1), or after documentation of a molecular leukemic relapse (Cohort 2)provided that research participants have tapered off steroids and haveno more than grade 2 acute graft-versus-host disease. Low-dosesubcutaneously administered IL-2 is given after the third T cellinfusion to support the in vivo persistence of transferred CTL. IL-2administration begins 24-hrs following adoptive transfer of T cellclones and continue for 14 days provided that no grade 3-4 toxicity (seebelow) is observed with the administration of the first two T cell dosesand that AGVHD is ≦grade 2. TABLE 2 CD19R⁺HyTK⁺, CD8⁺ Cytotoxic T CellAdministration Schedule Protocol Cell Dose Day Cell Dose I    0 1 × 10⁹cells/m² BSA II +14 5 × 10⁹ cells/m² BSA III +28 5 × 10⁹ cells/m² BSAwith s.c. IL-2 (5 × 10⁵ U/m2/dose q 12-hrs)

[0160] Each infusion consists of a composite of up to five T cell clonesto achieve the cell dose under study.

[0161] Study subjects who have B cell engraftment at the time relapse isdetected with CD20⁺ cells accounting for >10% of lymphocytes in thecirculation receive a single dose (250 mg/m²) of Rituximab (chimericanti-CD20 antibody) one week prior to the first T cell infusion.

[0162] On the day of infusion, T cell clones expanded in CRB-3008 areaseptically processed per standard technique on a CS-3000 bloodseparation device for cell washing and concentrating. Processed cellsare resuspended in 100 ml of 0.9% NaCl with 2% human serum albumin in abag for suitable for clinical re-infusion.

[0163] Study subjects are admitted to the GCRC at COHNMC for their Tcell infusions and are discharged no sooner than 23 hours followingtheir infusion provided that no toxicities are observed. Otherwisepatients remain hospitalized until resolution of any infusion-relatedtoxicity deemed to pose a significant risk to the study subject as anoutpatient.

[0164] T cells are infused intravenously over 30 minutes through acentral line if available, if not an age appropriate sized I.V. catheteris inserted into a peripheral vein. The I.V. tubing does not have afilter to avoid trapping of cells. The infusion bag is gently mixedevery 5 minutes during the infusion.

[0165] The doctor or his representative is present during the infusionand immediately available for 2 hours following the infusion. Nursingobservation and care is employed throughout the patient's hospital stay.

[0166] Subjects' oxygen saturation is measured by continuouspulse-oximetry beginning pre-infusion and continuing for at least 2 hrsor until readings return to their pre-infusion baseline.

[0167] Subjects experiencing transplant-related toxicities have theirinfusion schedule delayed until these toxicities have resolved. Thespecific toxicities warranting delay of T cell infusions include:

[0168] (a) Pulmonary: Requirement for supplemental oxygen to keepsaturation greater than 95% or presence of radiographic abnormalities onchest x-ray that are progressive; (b) Cardiac: New cardiac arrhythmianot controlled with medical management. Hypotension requiring pressorsupport; (c) Active Infection: Positive blood cultures for bacteria,fungus, or virus within 48-hours of day 0; (d) Hepatic: Serum totalbilirubin, or transaminases more than 5× normal limit; (e) Renal: Serumcreatinine >2.0 or if patient requires dialysis; (f) Neurologic: Seizureactivity within one week preceding day 0 or clinically detectableencephalopathy or new focal neurologic deficits; (g) Hematologic:Clinically evident bleeding diathesis or hemolysis.

[0169] Patients having anti-tumor responses based on bcr-abl Q-PCR butpersistent residual disease following the third T cell dose may haveadditional cell doses (5×10⁹ cells/m²/dose at 14 day intervals) withIL-2 (5×10⁵ U/m² q 12-hrs) provided grade 3 or higher toxicity isencountered.

[0170] b. Interleukin-2 Administration

[0171] Recombinant human IL-2 (rHuIL-2, Proleulin, Chiron, Emeryville,Calif.) resuspended for s.c. injection by standard pharmacy guidelinesis administered to study participants provided that (1) no grade 3-4toxicities are encountered at cell dose levels I-II and (2) GVHD is notmore than grade 2 off immunosuppressive medications. Based on previousexperience in Seattle administering s.c. IL-2 to melanoma patientsreceiving adoptive T cell therapy the IL-2 dose is 5×10⁵ U/m² q 12-hrsfor 14 days beginning on the day of T cell re-infusion #3.

[0172] c. Management of Toxcities and Complications

[0173] The management of mild transient symptoms such as have beenobserved with LAK, TIL, and T cell clone infusions symptoms is asfollows. (1) All patients are pre-medicated with 15 mg/kg ofacetaminophen p.o. (max. 650 mg.) and diphenhydramine 1 mg/kg I.V. (maxdose 50 mg). (2) Fever, chills and temperature elevations >101° F. aremanaged with additional tylenol as clinically indicated, 10 mg/kgibuprofen p.o. (max 400 mg) for breakthrough fevers, and 1 mg/kg demerolI.V. for chills (max 50 mg). Additional methods such as cooling blanketsare employed for fevers resistant to these measures. All subjects thatdevelop fever or chills have a blood culture drawn. Ceftriaxone 50 mg/kgI.V. (max dose 2 gms) is administered to non-allergic patients who inthe opinion of the physician in attendance appear septic; alternateantibiotic choices are used as clinically indicated. (3) Headache ismanaged with acetaminophen. (4) Nausea and vomiting are treated withdiphenhydramine 1 mg/kg I.V. (max 50 mg). (5) Transient hypotension isinitially managed by intravenous fluid administration, however, patientswith persistent hypotension require transfer to the intensive care unitfor definitive medical treatment. (6) Hypoxemia is managed withsupplemental oxygen.

[0174] Patients receive ganciclovir if grade 3 or 4 treatment-relatedtoxicity is observed. Parentally administered ganciclovir is dosed at 10mg/kg/day divided every 12 hours. A 14-day course is prescribed but maybe extended should symptomatic resolution not be achieved in that timeinterval. All patients not hospitalized at the time of presentingsymptoms are hospitalized for the first 72 hours of ganciclovir therapyfor monitoring purposes. If symptoms do not respond to ganciclovirwithin 72 hours additional immunosuppressive agents including but notlimited to corticosteroids and cyclosporin are added at the discretionof the principle investigator.

[0175] d. Concomitant Therapy

[0176] All standard supportive care measures for patients undergoingexperimental therapies are used at the discretion of the patient's Cityof Hope pediatric oncologist. Active infections occurring after studyenrollment are treated according to the standard of care. The followingagents are not allowed while on study: systemic cortico-steroids (exceptas outlined for management of T cell therapy toxicity), immunotherapy(for example—interferons, vaccines, other cellular products),pentoxifylline, or other investigational agents), ganciclovir or anyganciclovir derivatives for non-life threatening herpes virusinfections.

[0177] 4. Toxicities Monitored and Dosage Modifications

[0178] a. Toxicities to be Monitored

[0179] Toxicity criteria for the pilot phase I study is per the NCICommon Toxicity Criteria (CTC) version 2.0 for toxicity and AdverseEvent Reporting. A copy of the CTC version 2.0 is downloadable from theCTEP home page (http://ctep.info.nih.gov/l). All CTC guidelines apply totoxicity assessment except serum measurements of total bilirubin, ALTand AST. Due to the frequent and prolonged observed elevations inbilirubin and hepatic transaminases in cancer patients who have recentlyreceived chemotherapy, a grade 1 toxicity is an elevation from theirpre-T cell infusion base line up to 2.5× that baseline level. Grade 2hepatic is a >2.5-5× rise from their pre-T cell infusion baseline, agrade 3 toxicity >5-20× rise, and grade 4>20× baseline. Any toxicityreported by research participants while receiving treatment or infollow-up for which there is no specific CTC designation is graded onthe following scale: Grade 0- no toxicity, Grade 1- mild toxicity,usually transient, requiring no special treatment and generally notinterfering with usual daily activities, Grade 2- moderate toxicity thatmay be ameliorated by simple therapeutic maneuvers, and impairs usualactivities, Grade 3- severe toxicity which requires therapeuticintervention and interrupts usual activities. Hospitalization may berequired or may not be required. Grade 4- life-threatening toxicity thatrequires hospitalization.

[0180] b. Criteria for Dose Modification

[0181] If a patient develops grade 2 toxicity with dose level I, thesecond cell dose for that patient remains at T cell dose level I. Onlyif the maximal toxicity observed with the second infusion is limited tograde 2 will the third and final cell dose be administered. If the firstgrade 2 toxicity occurs with the second cell dose, the third cell doseis not be accompanied with s.c. IL-2.

[0182] c. Criteria for Removal of Patient from Treatment Regimen

[0183] If any patient develops grade 3 or higher toxicity or grade 3 orhigher GVHD, IL-2 if being administered is stopped. Ganciclovirtreatment as outlined above is initiated at the time a grade 3 or highertoxicity is encountered in those patients not receiving IL-2. For thosepatients receiving IL-2, ganciclovir treatment commences within 48-hoursof stopping IL-2 if the encountered toxicity has not decreased to ≦grade2 in that time interval. A grade 3 IL-2 injection site toxicity is anindication to discontinue IL-2 but not ablate T cells. Immunosuppressionfor GVHD is instituted in addition to ganciclovir administration inthose patients with grade 3 or higher GVHD. Any patient requiringganciclovir for T cell ablation does not receive further cell doses butcontinues being monitored per protocol. At the discretion of theprinciple investigator, corticosteroids and/or other immunosuppressivedrugs are added to ganciclovir should a more rapid tempo of resolutionof severe toxicities be indicated.

[0184] d. Research Participant Premature Discontinuation

[0185] Research participants who do not complete the study protocol areconsidered to have prematurely discontinued the study. The reasons forpremature discontinuation (for example, voluntary withdrawal, toxicity,death) are recorded on the case report form. Final study evaluations arecompleted at the time of discontinuation. Potential reasons forpremature discontinuation include: (a)the development of alife-threatening infection; (b) the judgment of the principalinvestigator that the patient is too ill to continue; (c) patient/familynoncompliance with study therapy and/or clinic appointments; (d)pregnancy; (e) voluntary withdrawal—a patient or his/her parents/legalguardians may remove himself/herself from the study at any time withoutprejudice; (f) significant and rapid progression of neuroblastomarequiring alternative medical, radiation or surgical intervention; (g)grade 3 or 4 toxicity judged to be possibly or probably related to studytherapy; and (h) technical difficulties are encountered in the T cellgenetic modification, cloning, and expansion procedure precluding thegeneration of clinical cell doses that meet all Quality Controlcriteria.

[0186] e. Study Closure

[0187] The study is discontinued if a grade 4 or higher toxicity is seenin the first two patients at dose level I or if at any time during theprotocol an incidence of grade 4 toxicity in study subjects exceeds 50%.Death from tumor progression greater than thirty days from the last Tcell infusion is not be scored as a grade V toxicity, nor be scored asan adverse event. The study can be terminated by the principalinvestigator, the IRB, or the Food and Drug Administration.

[0188] 5. Study Parameters and Calender (Table 3) TABLE 3 Calender ofSpecific Evaluations Infusion Infusion Screening Infusion Day + #2 Day +#3 Day + Day + Day + Day + Day + Visit #1 1 Day + 7 Day + 14 15 Day + 21Day + 28 29 35 42 56/70 100 History and X X X X X X X X X X X X XPhysical/ Lansky Score CBC, X X X X X X X X X X X X X diff, plt Chem X XX X X X X X X X X X X EBV, HIV X Serilogies Q-PCR X X X X X X X X X X XX for plasmid Sequence in PBMC Q-PCR for X X X X X X X X X BCR-ABL BoneMarrow X* X X Evaluation Morphologic/ Flow/ Chimerism Peripheral X Bloodfor Immune Response

[0189] To occur concurrently with the patient's evaluation for diseaserelapse and prior to commencing with salvage chemotherapy. The specificstudies/procedures include:

[0190] Review of pathologic specimens to confirm diagnosis of CD19⁺acute lymphoblastic leukemia.

[0191] Review molecular confirmation of Ph-positivity

[0192] Verify inclusion/exclusion criteria by history.

[0193] Administer the educational proctoring to the potential researchparticipant (≧7-yrs of age) and the parent/legal guardian, conduct thepost-educational assessment.

[0194] Obtain informed consent for enrollment from patient and donor.

[0195] Obtain EBV and HIV serologies.

[0196] For Ph⁻ patients in Cohort 1 ship a sample of blood/marrow to Dr.Radich (FHCRC) for generating leukemic clone PCR amplimers.

[0197] Conduct staging studies as outlined above.

[0198] Skin Biopsy from consented research participant for establishinga fibroblast cell line.

[0199] (b) Isolation of Peripheral Blood Mononuclear Cells For theInitiation of T Cell Cultures

[0200] Consented patients with HLA-matched related donors satisfyinginclusion/exclusion criteria undergo a leukapheresis procedure at theCity of Hope Donor/Apheresis Center. The leukapheresis product istransferred to CRB-3008 to initiate T cell cultures.

[0201] (c) Day -7 to -1: Pre-t Cell Infusion Restaging

[0202] Conduct restaging studies as outlined above.

[0203] Administration of Rituximab if peripheral CD20⁺ B cells accountfor more than 10% of circulating mononuclear cells.

[0204] (d) Day 0:evaluation immediately Prior to T Cell Infusion

[0205] Review of medical status and review of systems

[0206] Physical examination, vital signs, weight, height, body surfacearea

[0207] List of concomitant medications and transfusions

[0208] Karnofsky/Lansky performance status (see Table 4)

[0209] Complete blood count, differential, platelet count

[0210] Chem 18

[0211] Blood for protocol-specific (see Table 3) TABLE 4 Lansky Scale %Able to carry on 100 Fully active normal activity; 90 Minor restrictionin physically strenuous play no special care 80 Restricted in strenuousplay, tires more easily, needed otherwise active Mild to 70 Both greaterrestrictions of, and less time moderate spent in active play restriction60 Ambulatory up to 50% of time, limited active play withassistance/supervision 50 Considerable assistance required for anyactive play; fully able to engage in quiet play Moderate to 40 Able toinitiate quiet activities severe 30 Needs considerable assistance forquiet activity restriction 20 Limited to very passive activity initiatedby others (e.g. TV) 10 Completely disabled, not even passive play

[0212] (e) 0, +14, +28: Clinical Evaluation During and after T CellInfusions

[0213] Prior to the Infusion:

[0214] Interval History and Physical Exam

[0215] Blood draw for laboratory studies (see Table 3)

[0216] During the Infusion:

[0217] Vital signs at time 0, and every 15 minutes during the infusion,continuous pulse oximetery

[0218] Following the T Cell Infusion:

[0219] Vital Signs hourly for 12 hours

[0220] Oxygen saturation will be monitored for 2 hours following T cellinfusions. Values will be recorded prior to initiating the infusion,immediately post-infusion, and 2 hours post-infusion. In addition,values will be recorded every 15 minutes if they fall below 90% untilthe patient recovers to his/her pre-infusion room-air baselinesaturation.

[0221] Events will be managed by standard medical practice.

[0222] Prior to Discharge:

[0223] Interval History and Physical Exam

[0224] Blood draw for laboratory studies (see Table 3)

[0225] (f) Days +1, +7, +15, +21, +29, +35, +42, +56, +70, +100

[0226] Interval History and Physical Exam

[0227] Blood draw for CBC, diff, pit, and Chem 18

[0228] 3 cc/kg pt wt of heparinized (preservative-free heparin 10 U/10ml) blood sent to CRB-3002 for direct assay of peripheral bloodlymphocytes for vector DNA by PCR

[0229] (g) Bone Marrow Aspirate and Biopsy: Days −7−0, +56, +100

[0230] (h) If a research participant is taken off study after receivingT cells, restaging bone marrow evaluation will be evaluated 28 days and56 days following the last T cell dose administered.

[0231] 6. Criteria for Evaluation and Endpoint Definitions

[0232] (a) Criteria for Evaluation

[0233] The phase I data obtained at each clinical assessment is outlinedin Table 3. The following toxicity and adverse event determination willbe made: (a) symptoms and toxicities are evaluated as described above;(b) physical exam and blood chemistry/hematology results; and (c)adverse event reporting

[0234] (b) Disease Status

[0235] At each disease assessment outlined in Table 3 the determinationof measurable disease is recorded as follows: (1) Q-PCR forleukemic-specific amplimers or bcr-abl and (2) on days +56 and +100 bonemarrow studies will be evaluated and responses graded per standard ALLcriteria (Table 5). TABLE 5 Disease Response Criteria ProgressiveDisease >25% Increase in BCR-ABL Transcript (PD): By Q-PCR and/orProgression to Overt Relapse Stable Disease (SD): <25% Increase inBCR-ABL Signal by Q-PCR AND No Progression to Overt Relapse PartialResponse (PR): ≧25% Decrease in BCR-ABL Signal By Q-PCR AND No Evidenceof Overt Relapse Complete Response Loss of Detectable BCR-ABL Signal(CR): and No Evidence of Overt Relapse

[0236] 7. Reporting Adverse Events

[0237] Any sign, symptom or illness that appears to worsen during thestudy period regardless of the relationship to the study agent is anadverse event. All adverse events occurring during the study, whether ornot attributed to the study agent, that are observed by the Investigatoror reported by the patient are recorded on the Case Report Form and arereported as required by the FDA. Attributes include a description, onsetand resolution date, duration, maximum severity, assessment ofrelationship to the study agent or other suspect agent(s), action takenand outcome. Toxicities arising while on study are scored according to a0-4 scale based on the criteria delineated in the Common ToxicityCriteria (CTC) Version 2.0 (see above). Association or relatedness tothe study agent are graded as follows: 1=unrelated, 2=unlikely,3=possibly, 4=probably, and 5=definitely related.

[0238] Serious adverse events occurring during or after completion oftherapy are defined as any one of the following: (a) patient death,regardless of cause, occurring within 30 days of study agentadministration; (b) life threatening event; (c) prolongedhospitalization or requirement for additional hospitalizations duringtreatment and monitoring period due to toxicities attributed to study;(d) congenital anomaly in offspring conceived after initiation of study;(e) requirement for significant medical treatment due to toxicitiesencountered while on study; and (f) overdose of cells infused.

[0239] A life-threatening event is defined as having placed the patient,in the view of the Investigator, at immediate risk of death from theadverse event as it occurred. It does not include an adverse event that,had it occurred in a more serious form, might have caused death. Alladverse events that do not meet at least one of the above criteria aredefined as non-serious. Assessment of the cause of the event has nobearing on the assessment of the event's severity.

[0240] Unexpected adverse events are those which: (a) are not previouslyreported with adoptive T cell therapy and (b) are symptomatically andpathophysiologically related to a known toxicity but differ because ofgreater severity or specificity.

[0241] Appropriate clinical, diagnostic, and laboratory measures toattempt to delineate the cause of the adverse reaction in question mustbe performed and the results reported. All tests that reveal anabnormality considered to be related to adoptive transfer will berepeated at appropriate intervals until the course is determined or areturn to normal values occurs.

[0242] 8. Statistical Considerations

[0243] The considerations for the Phase I study of CD8⁺ cytotoxic Tcells genetically modified to express a CD19-specific chimericInimunoreceptor and HyTK for re-directed pre-B ALL targetingadministered to research participants who suffer a relapse of theirCD19⁺ ALL following HLA-matched allogeneic BMT are as follows. (a)Demographic and background characteristics obtained at enrollment arelisted and summarized. (b) The type and grade of toxicities noted duringtherapy are summarized for each dose level. (c) All adverse events notedby the investigator are tabulated according to the affected body system.(d) Descriptive statistics are used to summarize the changes frombaseline in clinical laboratory parameters. (e) For those patients withmeasurable tumor at the time T cell therapy commences, responses are bestratified per ALL response criteria (Table 5). (f) Kaplan-Meier productlimit methodology are used to estimate the survival. (g) 95% confidenceintervals are calculated for all described statistics.

[0244] It will be appreciated that the methods and compositions of theinstant invention can be incorporated in the form of a variety ofembodiments, only a few of which are disclosed herein. It will beapparent to the artisan that other embodiments exist and do not departfrom the spirit of the invention. Thus, the described embodiments areillustrative and should not be construed as restrictive.

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1 8 1 1931 DNA Artificial Sequence CD19R zeta chimeric receptor 1atctctagag ccgccacc atg ctt ctc ctg gtg aca agc ctt ctg ctc tgt 51 MetLeu Leu Leu Val Thr Ser Leu Leu Leu Cys 1 5 10 gag tta cca cac cca gcattc ctc ctg atc cca gac atc cag atg aca 99 Glu Leu Pro His Pro Ala PheLeu Leu Ile Pro Asp Ile Gln Met Thr 15 20 25 cag act aca tcc tcc ctg tctgcc tct ctg gga gac aga gtc acc atc 147 Gln Thr Thr Ser Ser Leu Ser AlaSer Leu Gly Asp Arg Val Thr Ile 30 35 40 agt tgc agg gca agt cag gac attagt aaa tat tta aat tgg tat cag 195 Ser Cys Arg Ala Ser Gln Asp Ile SerLys Tyr Leu Asn Trp Tyr Gln 45 50 55 cag aaa cca gat gga act gtt aaa ctcctg atc tac cat aca tca aga 243 Gln Lys Pro Asp Gly Thr Val Lys Leu LeuIle Tyr His Thr Ser Arg 60 65 70 75 tta cac tca gga gtc cca tca agg ttcagt ggc agt ggg tct gga aca 291 Leu His Ser Gly Val Pro Ser Arg Phe SerGly Ser Gly Ser Gly Thr 80 85 90 gat tat tct ctc acc att agc aac ctg gagcaa gaa gat att gcc act 339 Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu GlnGlu Asp Ile Ala Thr 95 100 105 tac ttt tgc caa cag ggt aat acg ctt ccgtac acg ttc gga ggg ggg 387 Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro TyrThr Phe Gly Gly Gly 110 115 120 act aag ttg gaa ata aca ggc tcc acc tctgga tcc ggc aag ccc gga 435 Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser GlySer Gly Lys Pro Gly 125 130 135 tct ggc gag gga tcc acc aag ggc gag gtgaaa ctg cag gag tca gga 483 Ser Gly Glu Gly Ser Thr Lys Gly Glu Val LysLeu Gln Glu Ser Gly 140 145 150 155 cct ggc ctg gtg gcg ccc tca cag agcctg tcc gtc aca tgc act gtc 531 Pro Gly Leu Val Ala Pro Ser Gln Ser LeuSer Val Thr Cys Thr Val 160 165 170 tca ggg gtc tca tta ccc gac tat ggtgta agc tgg att cgc cag cct 579 Ser Gly Val Ser Leu Pro Asp Tyr Gly ValSer Trp Ile Arg Gln Pro 175 180 185 cca cga aag ggt ctg gag tgg ctg ggagta ata tgg ggt agt gaa acc 627 Pro Arg Lys Gly Leu Glu Trp Leu Gly ValIle Trp Gly Ser Glu Thr 190 195 200 aca tac tat aat tca gct ctc aaa tccaga ctg acc atc atc aag gac 675 Thr Tyr Tyr Asn Ser Ala Leu Lys Ser ArgLeu Thr Ile Ile Lys Asp 205 210 215 aac tcc aag agc caa gtt ttc tta aaaatg aac agt ctg caa act gat 723 Asn Ser Lys Ser Gln Val Phe Leu Lys MetAsn Ser Leu Gln Thr Asp 220 225 230 235 gac aca gcc att tac tac tgt gccaaa cat tat tac tac ggt ggt agc 771 Asp Thr Ala Ile Tyr Tyr Cys Ala LysHis Tyr Tyr Tyr Gly Gly Ser 240 245 250 tat gct atg gac tac tgg ggt caagga acc tca gtc acc gtc tcc tca 819 Tyr Ala Met Asp Tyr Trp Gly Gln GlyThr Ser Val Thr Val Ser Ser 255 260 265 gta gaa ccc aaa tct tct gac aaaact cac acg tgc cca ccg tgc cca 867 Val Glu Pro Lys Ser Ser Asp Lys ThrHis Thr Cys Pro Pro Cys Pro 270 275 280 gca cct gaa ctc ctg ggg gga ccgtca gtc ttc ctc ttc ccc cca aaa 915 Ala Pro Glu Leu Leu Gly Gly Pro SerVal Phe Leu Phe Pro Pro Lys 285 290 295 ccc aag gac acc ctc atg atc tcccgg acc cct gag gtc aca tgc gtg 963 Pro Lys Asp Thr Leu Met Ile Ser ArgThr Pro Glu Val Thr Cys Val 300 305 310 315 gtg gtg gac gtg agc cac gaagac cct gag gtc aag ttc aac tgg tac 1011 Val Val Asp Val Ser His Glu AspPro Glu Val Lys Phe Asn Trp Tyr 320 325 330 gtg gac ggc gtg gag gtg cataat gcc aag aca aag ccg cgg gag gag 1059 Val Asp Gly Val Glu Val His AsnAla Lys Thr Lys Pro Arg Glu Glu 335 340 345 cag tac aac agc acg tac cgtgtg gtc agc gtc ctc acc gtc ctg cac 1107 Gln Tyr Asn Ser Thr Tyr Arg ValVal Ser Val Leu Thr Val Leu His 350 355 360 cag gac tgg ctg aat ggc aaggag tac aag tgc aag gtc tcc aac aaa 1155 Gln Asp Trp Leu Asn Gly Lys GluTyr Lys Cys Lys Val Ser Asn Lys 365 370 375 gcc ctc cca gcc ccc atc gagaaa acc atc tcc aaa gcc aaa ggg cag 1203 Ala Leu Pro Ala Pro Ile Glu LysThr Ile Ser Lys Ala Lys Gly Gln 380 385 390 395 ccc cga gaa cca cag gtgtac acc ctg cca cca tca cga gat gag ctg 1251 Pro Arg Glu Pro Gln Val TyrThr Leu Pro Pro Ser Arg Asp Glu Leu 400 405 410 acc aag aac cag gtc agcctg acc tgc ctg gtc aaa ggc ttc tat ccc 1299 Thr Lys Asn Gln Val Ser LeuThr Cys Leu Val Lys Gly Phe Tyr Pro 415 420 425 agc gac atc gcc gtg gagtgg gag agc aat ggg cag ccg gag aac aac 1347 Ser Asp Ile Ala Val Glu TrpGlu Ser Asn Gly Gln Pro Glu Asn Asn 430 435 440 tac aag acc acg cct cccgtg ctg gac tcc gac ggc tcc ttc ttc ctc 1395 Tyr Lys Thr Thr Pro Pro ValLeu Asp Ser Asp Gly Ser Phe Phe Leu 445 450 455 tac agc aag ctc acc gtggac aag agc agg tgg cag cag ggg aac gtc 1443 Tyr Ser Lys Leu Thr Val AspLys Ser Arg Trp Gln Gln Gly Asn Val 460 465 470 475 ttc tca tgc tcc gtgatg cat gag gct ctg cac aac cac tac acg cag 1491 Phe Ser Cys Ser Val MetHis Glu Ala Leu His Asn His Tyr Thr Gln 480 485 490 aag agc ctc tcc ctgtct ccc ggg aaa atg gcc ctg att gtg ctg ggg 1539 Lys Ser Leu Ser Leu SerPro Gly Lys Met Ala Leu Ile Val Leu Gly 495 500 505 ggc gtc gcc ggc ctcctg ctt ttc att ggg cta ggc atc ttc ttc aga 1587 Gly Val Ala Gly Leu LeuLeu Phe Ile Gly Leu Gly Ile Phe Phe Arg 510 515 520 gtg aag ttc agc aggagc gca gac gcc ccc gcg tac cag cag ggc cag 1635 Val Lys Phe Ser Arg SerAla Asp Ala Pro Ala Tyr Gln Gln Gly Gln 525 530 535 aac cag ctc tat aacgag ctc aat cta gga cga aga gag gag tac gat 1683 Asn Gln Leu Tyr Asn GluLeu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 540 545 550 555 gtt ttg gac aagaga cgt ggc cgg gac cct gag atg ggg gga aag ccg 1731 Val Leu Asp Lys ArgArg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 560 565 570 aga agg aag aaccct cag gaa ggc ctg tac aat gaa ctg cag aaa gat 1779 Arg Arg Lys Asn ProGln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 575 580 585 aag atg gcg gaggcc tac agt gag att ggg atg aaa ggc gag cgc cgg 1827 Lys Met Ala Glu AlaTyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 590 595 600 agg ggc aag gggcac gat ggc ctt tac cag ggt ctc agt aca gcc acc 1875 Arg Gly Lys Gly HisAsp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 605 610 615 aag gac acc tacgac gcc ctt cac atg cag gcc ctg ccc cct cgc 1920 Lys Asp Thr Tyr Asp AlaLeu His Met Gln Ala Leu Pro Pro Arg 620 625 630 taagcggccg c 1931 2 634PRT Artificial Sequence CD19R zeta chimeric receptor 2 Met Leu Leu LeuVal Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe LeuLeu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser 20 25 30 Leu Ser AlaSer Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser 35 40 45 Gln Asp IleSer Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly 50 55 60 Thr Val LysLeu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val 65 70 75 80 Pro SerArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr 85 90 95 Ile SerAsn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln 100 105 110 GlyAsn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 130 135140 Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala 145150 155 160 Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val SerLeu 165 170 175 Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg LysGly Leu 180 185 190 Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr TyrTyr Asn Ser 195 200 205 Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp AsnSer Lys Ser Gln 210 215 220 Val Phe Leu Lys Met Asn Ser Leu Gln Thr AspAsp Thr Ala Ile Tyr 225 230 235 240 Tyr Cys Ala Lys His Tyr Tyr Tyr GlyGly Ser Tyr Ala Met Asp Tyr 245 250 255 Trp Gly Gln Gly Thr Ser Val ThrVal Ser Ser Val Glu Pro Lys Ser 260 265 270 Ser Asp Lys Thr His Thr CysPro Pro Cys Pro Ala Pro Glu Leu Leu 275 280 285 Gly Gly Pro Ser Val PheLeu Phe Pro Pro Lys Pro Lys Asp Thr Leu 290 295 300 Met Ile Ser Arg ThrPro Glu Val Thr Cys Val Val Val Asp Val Ser 305 310 315 320 His Glu AspPro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 325 330 335 Val HisAsn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 340 345 350 TyrArg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 355 360 365Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 370 375380 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 385390 395 400 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn GlnVal 405 410 415 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp IleAla Val 420 425 430 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr LysThr Thr Pro 435 440 445 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu TyrSer Lys Leu Thr 450 455 460 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn ValPhe Ser Cys Ser Val 465 470 475 480 Met His Glu Ala Leu His Asn His TyrThr Gln Lys Ser Leu Ser Leu 485 490 495 Ser Pro Gly Lys Met Ala Leu IleVal Leu Gly Gly Val Ala Gly Leu 500 505 510 Leu Leu Phe Ile Gly Leu GlyIle Phe Phe Arg Val Lys Phe Ser Arg 515 520 525 Ser Ala Asp Ala Pro AlaTyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 530 535 540 Glu Leu Asn Leu GlyArg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 545 550 555 560 Arg Gly ArgAsp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 565 570 575 Gln GluGly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 580 585 590 TyrSer Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 595 600 605Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 610 615620 Ala Leu His Met Gln Ala Leu Pro Pro Arg 625 630 3 29 DNA ArtificialSequence 5′ scFvFc PCR primer 3 tcttcctcta cacagcaagc tcaccgtgg 29 4 24DNA Artificial Sequence 3′ human zeta chain PCR primer 4 gagggttcttccttctcggc tttc 24 5 25 DNA Artificial Sequence 5′FAM-3′ TAMRA labeledhybridization probe 5 ttcactctga agaagatgcc tagcc 25 6 20 DNA ArtificialSequence human betaglobin 5′PCR primer 6 acacaactgt gttcactagc 20 7 20DNA Artificial Sequence human betaglobin 3′ PCR primer 7 gtctccttaaacctgtcttg 20 8 23 DNA Artificial Sequence 5′ HEX-3′ TAMRA labeledhybridization probe 8 acctgactcc tgaggagaag tct 23

What is claimed is:
 1. Genetically engineered CD19-specific immune cellswhich express and bear on the cell surface membrane a CD19-specificchimeric receptor, wherein the chimeric receptor consists of anintracellular signaling domain for an effector function of the immunecell, at least one transmembrane domain and at least one extracellulardomain, the extracellular domain comprising a CD19-specific receptor. 2.The CD19-specific immune cells of claim 1, wherein the immune cells areselected from the group consisting of T-cells, natural killer cells,macrophage, neutrophils and bone marrow stem cells.
 3. The CD19-specificimmune cells of claim 2 which are non-malignant human cells. 4.Genetically engineered CD19-specific T cells which express and bear onthe cell surface membrane a CD19-specific chimeric receptor, wherein thechimeric receptor consists of: a) an intracellular signaling domainselected from the group of intracellular signaling domains selected fromthe group consisting of the (1) zeta, eta, delta, gamma or epsilon chainof CD3, (2) MB1 chain, (3) B29, (4) FcγRIII and (5) FcεRI; b) at leastone transmembrane domain; and c) at least one extracellular domaincomprising a CD19-specific receptor.
 5. The CD19-specific T cells ofclaim 4 which are CD4⁺ and which produce IL-2 when co-cultured in vitrowith CD19⁺ malignant B cells.
 6. The CD19-specific T cells of claim 4which are CD8⁺ or CD4⁺ and which lyse CD19⁺ target malignant B-cellswhen co-cultured in vitro with the target cells.
 7. The CD19-specific Tcells of claim 4 which comprises a mixed population of CD4⁺ and CD8⁺cells.
 8. The CD19-specific T cells of claim 4 wherein the CD19-specificreceptor comprises the Fv region of a single chain monoclonal antibodyto CD19.
 9. The CD19-specific T-cells of claim 8 wherein theintracellular signaling domain is from the human CD3 zeta chain.
 10. TheCD19-specific T cells of claim 9 wherein the CD19-specific chimericreceptor comprises scFvFc:ζ, wherein scFvFc represents the extracellulardomain, scFv represents the V_(H) and V_(L) chains of a single chainmonoclonal antibody to CD19, Fc represents at least part of a constantregion of an IgG₁, and ζ represents the intracellular signaling domainofthe zeta chain of human CD3.
 11. The CD19-specific T cells of claim 10wherein the extracellular domain and the intracellular signaling domainare linked by the transmembrane domain of human CD4.
 12. TheCD19-specific T cells of claim 10 wherein the chimeric receptor is aminoacids 23-634 of SEQ ID NO:2.
 13. A CD19-specific chimeric T cellreceptor consisting of: a) an intracellular signaling domain selectedfrom the group of intracellular signaling domains selected from thegroup consisting of the (1) zeta, eta, delta, gamma or epsilon chain ofCD3, (2) MB1 chain, (3) B29, (4) FcγRIII and (5) FcεRI; b) at least onetransmembrane domain; and c) at least one extracellular domaincomprising a CD19-specific receptor.
 14. The CD19-specific chimeric Tcell receptor of claim 13 that is a chimeric T cell receptor whichcomprises scFvFc:ζ, wherein scFvFc represents the extracellular domain,scFv represents the V_(H) and V_(L) chains of a single chain monoclonalantibody to CD19, Fc represents at least part of a constant region of anIgG₁, and ζ represents the effector function intracellular signalingdomain of the zeta chain of human CD3.
 15. The CD19-specific chimeric Tcell receptor of claim 14 wherein the extracellular domain and theintracellular domain are linked by the transmembrane domain of humanCD4.
 16. The CD19-specific chimeric T cell receptor of claim 15 which isamino acids 23-634 of SEQ ID NO:2.
 17. A DNA construct encoding aCD19-specific chimeric T cell receptor of any one of claims 13-16.
 18. Aplasmid expression vector containing a DNA construct of claim 17 inproper orientation for expression.
 19. A method of treating a CD19⁺malignancy in a mammal which comprises infusing into the animalCD19-specific immune cells of claim 1 in a therapeutically effectiveamount.
 20. A method of treating a CD19⁺ malignancy in a human patientwhich comprises infusing into the patient human CD19-specific T cells ofany of claims 4 through 12 in a therapeutically effective amount andoptionally contemporaneously administering to the patient IL-2 in anamount effective to augment the effect of the T cells.
 21. The method ofclaim 19 or claim 20 where the malignancy is selected from the groupconsisting of CD19⁺ acute lymphoblastic leukemia, chronic lymphoblasticleukemia, CD19+ lymphoma and chronic lymphocytic leukemia.
 22. Themethod of claim 19 or 20 wherein the patient has previously undergonemyeloablative chemotherapy and stem cell rescue.
 23. A method of makingand expanding the CD19-specific T cells of claim 4 which comprisestransfecting T cells with an expression vector containing a DNAconstruct encoding the CD19-specific chimeric receptor, then stimulatingthe cells with CD19⁺ cells, recombinant CD19, or an antibody to thechimeric receptor to cause the cells to proliferate.
 24. The method ofclaim 23 wherein the DNA has been depleted of endotoxin andelectroporation occurs after the cells have been stimulated with amitogen.
 25. The method of claim 24 wherein the T cells arenon-malignant human cells.
 26. The method of claim 25 wherein the Tcells are peripheral blood mononuclear cells.
 27. The method of claim 23wherein the intracellular signaling domain of the chimeric receptor isthe zeta chain of human CD3.
 28. The CD19-specific T cells of claim 9wherein the CD19-specific chimeric receptor comprises scFvFc:ζ, whereinscFvFc represents the extracellular domain, scFv represents the V_(H)and V_(L) chains of a single chain monoclonal antibody to CD19, Fcrepresents at least part of a constant region of an IgG₁, and ζrepresents the intracellular signaling domain of the zeta chain of humanCD3.
 29. The method of claim 28 wherein the wherein the extracellulardomain and the intracellular signaling domain are linked by thetransmembrane domain of human CD4.
 30. The method of claim 29 whereinthe chimeric receptoris amino acids 23-634 of SEQ ID NO:2.
 31. Themethod of any of claims 23-30 wherein the transfected cells are clonedand a clone demonstrating presence of a single integrated unrearrangedplasmid and expression of the chimeric receptor is expanded ex vivo. 32.The method of claim 31 wherein the clone selected for ex vivo expansionis CD8⁺ and demonstrates the capacity to specifically recognize and lyseCD19⁺ target cells.
 33. The method of claim 32 wherein the cloneselected for ex vivo expansion demonstrates an enhanced capacity tospecifically recognize and lyse CD19⁺ target cells when compared toother cells transfected in the same manner.
 34. The method of claim 33wherein the chimeric receptor comprises an scFvFc:ζ receptor and theclone is expanded by stimulation with IL-2 and OKT3 antibody.
 35. Amethod of abrogating an untoward B cell function in a patient whichcomprises administering to the patient CD19-specific T cells of claim 4in a therapeutically effective amount.
 36. The method of claim 35wherein the CD19-specific T cells are administered to treat anautoimmune disease in the patient.
 37. The method of claim 36 whereinthe autoimmune disease is mediated in whole or in part by B-cells. 38.The method of claim 37 wherein the CD19-specific redirected T cells areadministered to produce immunosuppression in the patient prior toadministering a foreign substance to the patient.
 39. The method ofclaim 38 wherein the foreign substance is a monoclonal antibody, DNA, avirus or a cell.